Compositions and methods for regulating bacterial pathogenesis

ABSTRACT

The production of a purified extracellular bacterial signal called autoinducer-2 is regulated by changes in environmental conditions associated with a shift from a free-living existence to a colonizing or pathogenic existence in a host organism. Autoinducer-2 stimulates LuxQ luminescence genes, and is believed also to stimulate a variety of pathogenesis related genes in the bacterial species that produce it. A new class of bacterial genes is involved in the biosynthesis of autoinducer-2.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/453,976, filed Dec. 2, 1999, which claims priority from U.S.Provisional Application Ser. No. 60/110,570, filed Dec. 2, 1998, both ofwhich are incorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Science Foundation, GrantNo. MCB-9506033.

FIELD OF THE INVENTION

This invention relates to the field of bacterial diseases of humans andother mammals. In particular, the invention provides novel genes andsignaling factors involved in inducing pathogenesis in certain bacteria,and methods for controlling such pathogenesis through manipulation ofthose factors and genes.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application to more fullydescribe the state of the art to which this invention pertains. Thedisclosure of each such publication is incorporated by reference herein.

The control of gene expression in response to cell density, or quorumsensing, was first described in the marine luminous bacteria Vibriofischeri and Vibrio harveyi. This phenomenon has recently becomerecognized as a general mechanism for gene regulation in many Gramnegative bacteria. Quorum sensing bacteria synthesize, release, andrespond to specific acyl-homoserine lactone signaling molecules calledautoinducers to control gene expression as a function of cell density.In all acyl-homoserine lactone quorum sensing systems described to date,except that of V. harveyi, the autoinducer synthase is encoded by a genehomologous to luxI of V. fischeri, and response to the autoinducer ismediated by a transcriptional activator protein encoded by a genehomologous to luxR of V. fischeri (Bassler and Silverman, in Twocomponent Signal Transduction, Hoch et al., eds, Am. Soc. Microbiol.Washington D.C., pp 431-435, 1995). In contrast, V. harveyi has twoindependent density sensing systems (called Signaling Systems 1 and 2),and each is composed of a sensor-autoinducer pair. V. harveyi SignalingSystem 1 is composed of Sensor 1 and autoinducer 1 (AI-1), and thisautoinducer is N-(3-hydroxybutanoyl)-L-homoserine lactone (see Bassleret al, Mol. Microbiol. 9: 773-786, 1993). V. harveyi Signaling System 2is composed of Sensor 2 and autoinducer 2 (AI-2) (Bassler et al., Mol.Microbiol. 13: 273-286, 1994). The structure of AI-2 heretofore has notbeen determined, nor have the gene(s) involved in biosynthesis of AI-2been identified. Signaling System 1 is a highly specific system proposedto be used for intra-species communication and Signaling System 2appears to be less species-selective, and is hypothesized to be forinter-species communication (Bassler et al., J. Bacteriol. 179:4043-4045, 1997).

Reporter strains of V. harveyi have been constructed that are capable ofproducing light exclusively in response to AI-1 or to AI-2 (Bassler etal., 1993, supra; Bassler et al., 1994, supra). V. harveyi reporterstrains have been used to demonstrate that a few species of bacteriaproduce stimulatory substances that mimic the action of AI-2 (Bassler etal., 1997, supra).

Quorum sensing in V. harveyi, mediated by Signaling Systems 1 and 2,triggers the organisms to bioluminesce at a certain cell density. Thesesame signaling systems, particularly Signaling System 2, are believed totrigger other physiological changes in V. harveyi and other bacteriapossessing the same signaling system. Thus, it would be an advance inthe art to identify and characterize the signaling factor autoinducer-2and the genes encoding the proteins required for its production. Such anadvance would provide a means to identify a novel class of compoundsuseful for controlling mammalian enteric or pathogenic bacteria.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been discoveredthat a variety of bacterial species, some of them mammalian pathogens,secrete an organic signaling molecule that stimulates the expression ofluminescence in the V. harveyi Signaling System 2 bioassay. The moleculesecreted by these organisms mimics V. harveyi AI-2 in its physical andfunctional features. The production in bacteria of this novel signalingmolecule is regulated by changes in environmental conditions associatedwith a shift from a free-living existence to a colonizing or pathogenicexistence in a host organism. Thus, in addition to stimulatingluminescence genes (specifically luxCDABE) in V. harveyi, the signalingmolecule is expected to stimulate a variety of pathogenesis relatedgenes in the bacterial species that produce it. A highly purified formof the signaling molecule is provided in the present invention. Alsoprovided is a new class of bacterial genes involved in the biosynthesisof the signaling molecule.

According to one aspect, the present invention provides an isolatedbacterial extracellular signaling factor comprising at least onemolecule that is polar and uncharged, and having an approximatemolecular weight of less than 1,000 kDa, wherein said factor interactswith LuxQ protein thereby inducing expression of a Vibrio harveyi operoncomprising luminescence genes luxCDABE. In a preferred embodiment, thefactor possesses a specific activity wherein about 0.1 to 1.0 mg of apreparation of the factor stimulates about a 1,000-fold increase inluminescense, as measured in a bioassay using a V. harveyi Sensor 2+reporter strain. In a particularly preferred embodiment, the factor ispurified in such a way that it possesses a specific activity whereinabout 1 to 10 μg of a preparation of the factor stimulates about a1,000-fold increase in luminescence, as measured in a bioassay using aV. harveyi Sensor 2+ reporter strain.

The signaling factor of the invention is produced by a variety ofbacteria, including but not limited to: Vibrio harveyi, Vibrio cholerae,Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum,Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium,Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borreliaburgfdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersiniapestis, Campylobacter jejuni, Deinococcus radiodurans, Mycobacteriumtuberculosis, Enterococcus faecalis, Streptococcus pneumoniae,Streptococcus pyogenes and Staphylococcus aureus.

In another aspect, the invention provides an isolated bacterialsignaling factor having the formula:

In another aspect, the invention provides a method for identifying acompound that regulates the activity of a signaling factor by contactingthe signaling factor with the compound, measuring the activity of thesignaling factor in the presence of the compound and comparing theactivity of the signaling factor obtained in the presence of thecompound to the activity of the signaling factor obtained in the absenceof the compound and identifying a compound that regulates the activityof the signaling factor.

In yet another aspect, the invention provides a method for detecting anautoinducer molecule in a sample by contacting the sample with abacterial cell, or extract thereof, comprising biosynthetic pathwaysthat produce a detectable amount of light in response to an exogenousautoinducer, the bacterial cell having at least two distinct alterationsin gene loci that participate in autoinducer pathways, wherein a firstalteration in a gene locus comprises an alteration that inhibitsdetection of a first autoinducer and wherein a second alteration in agene locus comprises an alteration that inhibits production of a secondautoinducer and measuring light produced by the bacterial cell, orextract thereof.

In another aspect, the invention provides a bacterial cell having atleast two distinct alterations in gene loci that participate inautoinducer pathways, wherein a first alteration in a gene locuscomprises an alteration that inhibits detection of a first autoinducerand wherein a second alteration in a gene locus comprises an alterationthat inhibits production of a second autoinducer and wherein the cell isbioluminescent when contacted with an autoinducer.

In another aspect, the invention provides a method for identifying anautoinducer analog that regulates the activity of an autoinducer bycontacting a bacterial cell, or extract thereof, comprising biosyntheticpathways which will produce a detectable amount of light in response toan autoinducer with an autoinducer analog and comparing the amount oflight produced by the bacterial cell, or extract thereof, in thepresence of an autoinducer with the amount produced in the presence ofthe autoinducer analog, wherein a change in the production of light isindicative of an autoinducer analog that regulates the activity of anautoinducer.

In another aspect, the invention provides a method for producingautoinducer-2 by contacting S-adenosylhomo-cysteine (SAH) with a LuxSprotein under conditions and for such time as to promote the conversionof S-adenosylhomo-cysteine to autoinducer-2.

In another aspect, the invention provides a method for producingautoinducer-2 by contacting S-ribosylhomo-cysteine (SRH) with a LuxSprotein under conditions and for such time as to promote the conversionof S-ribosylhomocysteine to autoinducer-2.

In another aspect, the invention provides A method for producingautoinducer-2 by contacting S-adenosylhomo-cysteine (SAH) with a5′-methylthioadenosine/S-adenosylhomo-cysteine nucleosidase proteinunder conditions and for such time as to promote the conversion ofS-adenosylhomocysteine to S-ribosylhomocysteine; contacting theabove-described S-ribosylhomocysteine with a LuxS protein underconditions and for such time as to promote the conversion ofS-ribosylhomocysteine to autoinducer-2.

In another aspect, the invention provides a method for detecting anautoinducer-associated bacterial biomarker by contacting at least onebacterial cell with an autoinducer molecule under conditions and forsuch time as to promote induction of a bacterial biomarker and detectingthe bacterial biomarker.

In another aspect, the invention provides a method for detecting atarget compound that binds to a LuxP protein by contacting the LuxPprotein with the target compound and detecting binding of the compoundto LuxP.

In another aspect, the invention provides a method for regulatingbacterial biofilm formation comprising contacting a bacterium capable ofbiofilm formation with a compound capable of regulating biofilmformation, wherein the compound regulates autoinducer-2 activity.

According to another aspect of the invention, a method is provided forpurifying the aforementioned bacterial extracellular signaling factor.The method comprises the steps of: (a) growing, in a culture medium,bacterial cells that produce the signaling molecule; (b) separating thebacterial cells from the culture medium; (c) incubating the bacterialcells in a solution having high osmolarity, under conditions that permitproduction and secretion of the signaling molecule from the bacterialcells; (d) separating the bacterial cells from the high osmolaritysolution; and (e) purifying the factor from the high osmolaritysolution. The method may further comprise: (f) separating polar factorsfrom non-polar factors in an evaporated sample of the high osmolaritysolution; and (g) subjecting the polar factors to reverse-phase HighPerformance Liquid Chromatography. In a preferred embodiment, the highosmolarity solution comprises at least 0.4 M monovalent salt, mostpreferably 0.4-0.5 M NaCl.

In another preferred embodiment, the method further comprises growingthe bacterial cells in a culture medium containing a carbohydrateselected from the group consisting of glucose, fructose, mannose,glucitol, glucosamine, galactose and arabinose.

According to another aspect of the invention, an isolated nucleic acidmolecule is provided, which encodes a protein necessary for biosynthesisof a bacterial extracellular signaling factor that induces expression ofa Vibrio harveyi LuxQ luminescence gene. The nucleic acid molecule maybe isolated from a wide variety of bacteria, including but not limitedto: Vibrio harveyi, Vibrio cholera, Salmonella typhimurium, Escherichiacoli, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis andBorrelia burdorferi.

The aforementioned nucleic acid molecule encodes a protein havingbetween about 150 and 200 amino acid residues. Preferably, the encodedprotein comprises an amino acid sequence substantially the same as asequence selected from the group consisting of any of SEQ ID NOS:10-17,or a consensus sequence derived from a comparison of two or more of SEQID NOS: 10-17. The nucleic acid molecule preferably has a sequencesubstantially the same as a sequence selected from the group consistingof any of SEQ ID NOS:1-9, or a consensus sequence derived from acomparison of two or more of SEQ ID NOS: 1-9.

Recombinant DNA molecules comprising the aforementioned nucleic acidmolecules are also provided in accordance with the present invention, aswell as proteins produced by expression of any of the nucleic acidmolecules.

Additional features and advantages of the present invention will bebetter understood by reference to the drawings, detailed description andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Signaling substance from E. coli AB1157 and S. typhimurium LT2cell-free culture fluids that induces luminescence in V. harveyi. Theresponses of V. harveyi reporter strains BB170 (Sensor 1⁻, Sensor 2⁺)(FIG. 1A), and BB886 (Sensor 1⁺, Sensor 2⁻) (FIG. 1B) to signalingsubstances present in cell-free culture fluids from E. coli, S.typhimurium and V. harveyi strains are shown. A bright culture of eachreporter strain was diluted 1:5000 into fresh medium, and the lightproduction per cell was then measured during the growth of the dilutedculture. Cell-free culture fluids or sterile growth medium were added ata final concentration of 10% (v/v) at the start of the experiment. Thedata for the 5 hour time point are shown and are presented as thepercent of the activity obtained when V. harveyi cell-free spent culturefluids are added. Abbreviations used for the different strains are: V.h;Vibrio harveyi, S.t; Salmonella typhimurium, and E.c; Escherichia coli.

FIG. 2. Active secretion of the signaling molecule by viable E. coli andS. typhimurium. The response of the V. harveyi reporter strain BB170(Sensor 1⁻, Sensor 2⁺) to a signaling substance produced and secreted byE. coli AB1157 and S. typhimurium LT2 but not E. coli DH5 is shown. V.harveyi reporter strain BB170 was diluted 1:5000 in AB medium and lightoutput per cell was monitored during growth. At the start of theexperiment, either 1×10⁶ E. coli AB1157, S. typhimurium LT2 or E. coliDH5 washed and resuspended viable cells (left-hand, white bars) orUV-killed cells (right-hand, black bars) was added. The data arepresented as the fold-activation above the endogenous level ofluminescence expressed by V. harveyi BB170 at the 5 hour time point.Abbreviations used for the different strains are: S.t; Salmonellatyphimurium, and E.c; Escherichia coli.

FIG. 3. Effect of glucose depletion on the production and degradation ofthe signaling activity by S. typhimurium LT2. S. typhimurium LT2 wasgrown in LB medium containing either 0.1% glucose (FIG. 3A) or 0.5%glucose (FIG. 3B). At the specified times cell-free culture fluids wereprepared and assayed for signaling activity in the luminescencestimulation assay (Bars), and the concentration of glucose remaining(circles). The cell number was determined at each time by diluting andplating the S. typhimurium LT2 on LB medium and counting colonies thenext day (squares). The signaling activity is presented as the percentof the activity obtained when V. harveyi cell-free spent culture fluidsare added. These data correspond to the 5 h time point in theluminescence stimulation assay. The glucose concentration is shown as %glucose remaining. Cell number is cells/ml×10⁻⁹. The symbol \\ indicatesthat the time axis is not drawn to scale after 8 h.

FIG. 4. Response curve of V. harveyi to AI-2 produced by V. harveyi andS. typhimurium. The V. harveyi reporter strain BB170 (Sensor 1⁻, Sensor2⁺) was tested for its response to the addition of exogenous AI-2 madeby V. harveyi strain BB152 (AI-1⁻, AI-2⁺) and to that made by S.typhimurium LT2. A bright culture of the reporter strain was diluted1:5000 and either 10% (v/v) growth medium (closed circles), cell-freeculture fluid from V. harveyi BB152 grown overnight in AB (opencircles), or cell-free culture fluid from S. typhimurium LT2 grown for 6h on LB+0.5% glucose (closed squares) was added at the start of theexperiment. RLU denotes relative light units and is defined as (countsmin⁻¹×10³)/(colony-forming units ml⁻¹).

FIG. 5. Conditions affecting autoinducer production in S. typhimurium.S. typhimurium LT2 was subjected to a variety of treatments after whichcell-free culture fluids or osmotic shock fluids were prepared. Thesepreparations were added to a diluted culture of the V. harveyi AI-2reporter strain BB170 at 10% (v/v) and light output was measuredthereafter. Fold activation is the level of light produced by thereporter following addition of the specified S. typhimurium preparationdivided by the light output of the reporter when growth medium alone wasadded. The bars in FIG. 5A represent cell-free fluids prepared from S.typhimurium after the following treatments: LB 6 h; 6 h growth in LB at30° C., LB+Glc 6 h; 6 h growth in LB+0.5% glucose at 30° C., LB+Glc 24h; 24 h growth in LB+0.5% glucose at 30° C. In all the experimentspresented in FIG. 5B, the S. typhimurium were pre-grown at 30° C. for 6h in LB containing 0.5% glucose, then pelleted and resuspended for 2 hunder the following conditions: LB; in LB at 30° C., LB+Glc; in LB+0.5%glucose at 30° C., LB pH 5; in LB at pH 5.0 at 30° C., 0.4 M NaCl; in0.4 M NaCl at 30° C., 0.1 M NaCl; in 0.1 M NaCl at 30° C., and HeatShock 43°; in LB+0.5% glucose at 43° C. After these two hour treatments,cell-free fluids were prepared from each sample and assayed.

FIG. 6. S. typhimurium signaling activity in limiting and non-limitingconcentrations of glucose. S. typhimurium LT2 was grown in LB in thepresence of limiting (0.1%) and non-limiting (1.0%) concentrations ofglucose. The activity present in the cell-free culture fluids (blackbars) was assayed at the times indicated and normalized to that producedby 1×10⁹ cells. The increase in signaling activity measured in the 0.4 MNaCl osmotic shock fluids prepared from the same cells is shown as thewhite bars on top of the black bars. These data are also normalized for1×10⁹ cells. The signaling activity for limiting glucose is shown inFIGS. 6A, 6C, and 6E, and that for non-limiting glucose is shown inFIGS. 6B, 6D, and 6F. FIGS. 6A and 6B also show the percent glucoseremaining (triangles), FIGS. 6C and 6D show the cell number (squares),and Panels E and F show the pH (circles) at each time point.

FIG. 7. Effects of glucose and pH on signal production by S.typhimurium. The quorum sensing signal released by S. typhimurium LT2was measured when the cells were grown in LB medium containing 0.5%glucose at pH 7.2 (FIG. 7A, bars), and when the cells were grown in LBat pH 5.0 without an added carbon source (FIG. 7B, bars). The level ofsignal present in cell free culture fluids (black bars) and in 0.4 MNaCl osmotic shock fluids was measured (white bars on top of black bars)at the time points indicated. In each panel, the circles represent thepH of the medium, and the squares show the cell number at the differenttime points.

FIG. 8. High osmolarity induces signal release and low osmolarityinduces signal degradation in S. typhimurium LT2. The quorum sensingsignal released by S. typhimurium LT2 resuspended in 0.4 M NaCl and in0.1 M NaCl was measured in the presence and absence of proteinsynthesis. S. typhimurium LT2 was pre-grown in LB containing 0.5%glucose for 6 h. The cells were harvested and resuspended in 0.4 M NaCl(FIG. 8A) or 0.1 M NaCl (FIG. 8B) in the presence or absence of 30 g/mlCm for the time periods indicated. In each panel, the open symbolsrepresent the activity measured in the absence of Cm and the closedsymbols represent the activity measured in the presence of Cm.

FIG. 9. The luxS and ygaG genes from V. harveyi and E. coli MG1655. FIG.9A shows a restriction map of the V. harveyi luxS_(V.h.) chromosomalregion which was defined by Tn5 insertion. The sites of Tn5 insertionsthat disrupted the AI-2 production function and one control Tn5insertion outside of the luxS_(V.h.) locus are shown (triangles). FIG.9B depicts the ygaG region in the E. coli MG1655 chromosome. This ORF isflanked by the emrB and gshA genes. The direction of transcription ofeach gene is indicated by the horizontal arrows. The correspondingposition of the MudJ insertion that eliminated AI-2 production in S.typhimurium LT2 is shown by a vertical arrow. H, R, P, and B denoteHindIII, EcoRI, PstI and BamHI restriction sites respectively.

FIG. 10. Autoinducer production phenotypes of V. harveyi and S.typhimurium strains. Cell-free culture fluids from V. harveyi and S.typhimurium strains were prepared and tested for AI-2 activity in the V.harveyi BB170 bioassay. FIG. 10A: AI-2 production phenotypes of the wildtype V. harveyi strain MM28 which contains a Tn5 insertion outside ofluxS_(V.h.) (denoted WT) and the luxS_(V.h) ::Tn5 mutant strain MM30(denoted luxS⁻). FIG. 10B: AI-2 production phenotypes of wild type S.typhimurium LT2 (denoted WT) and the ygaG::MudJ insertion mutant strainCS132 (denoted ygaG⁻). Activity is reported as fold-induction ofluminescence expression of the V. harveyi BB170 reporter strain overthat when sterile medium was added.

FIG. 11. Complementation of AI-2 production in S. typhimurium CS132 andE. coli DH5. Cell-free culture fluids from E. coli and S. typhimuriumstrains were tested for AI-2 activity in the bioassay. The activitypresent in these fluids was compared to that produced by wild type V.harveyi BB120. In the figure, the level of BB120 activity was normalizedto 100%. FIG. 11A: AI-2 activity in cell-free fluids from wild type V.harveyi BB120, E. coli O157:H7, and S. typhimurium LT2. FIG. 11B:Complementation of S. typhimurium CS132 (ygaG::MudJ) and FIG. 11C:Complementation of E. coli DH5. In Panel B and C, the in trans AI-2production genes are the following: vector control (denoted: none), E.coli O157:H7ygaG; and V. harveyi BB120 luxS_(V.h) . E. coli and V.harveyi are abbreviated E.c. and V.h. respectively.

FIG. 12. Alignment of LuxS and YgaG protein sequences. The translatedprotein sequences for the AI-2 production family of proteins are shown.We determined the sequences for the luxS_(V.h.) gene from V. harveyiBB120 (SEQ ID NO:10), and the ygaG genes (re-named herein as luxS_(E.C.)from E. coli MG1655 (SEQ ID NO: 11), E. coli O157:H7 (SEQ ID NO: 11),and E. coli DH5 (SEQ ID NO: 18). The S. typhimurium LT2 ygaG (re-namedherein luxS_(S.T.) partial sequence (SEQ ID NO: 12) came from the S.typhimurium database. Amino acid residues that are not identical to theLuxS_(V.H.) protein are underlined and not in bold font. The site of theframe shift mutation in the E. coli DH5 DNA sequence is denoted by an“*”. The 20 altered amino acid residues that are translated followingthe frame shift are enclosed by the box.

FIG. 13. A diagram of the hybrid quorum sensing circuit of Vibrioharveyi is provided. The AI-1 and AI-2 circuits are independentlystimulated but integrate their signals for light expression. Eachpathway, however, is also independently competent to generate light.This allows for reciprocal mutations in the LuxN or LuxQ sensors to beused to construct a reporter specific for AI-2 or AI-1, respectively.

FIG. 14. Response phenotypes of V. harveyi wild-type and lux regulatorymutants. At the first time point, cell-free culture fluids (10%), ornothing (N.A) was added. Wild-type, cell-free culture fluid (AI-1+AI2);LuxS⁻ cell-free culture fluid (AI-1); LuxM⁻ cell-free culture fluid(AI-2). Relative light units are defined as cpm×10³/CFU/ml.

FIG. 15. A diagram of the biosynthetic pathway of autoinducer-2 (AI-2),including the structure of AI-2, is shown.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have identified, isolatedand characterized an extracellular signaling factor produced by severalstrains of pathogenic bacteria, including Salmonella typhimurium andEscherichia coli, which has a role in regulating the pathogenesis orvirulence of these bacteria. We have also identified and cloned genesinvolved in the biosynthesis of this signaling factor. The purificationand/or cloning of this signaling molecule and the genes that encodeproteins that catalyze its biosynthesis open a new avenue for drugdesign aimed at either inhibition of production of or response to thismolecule by bacteria. Drugs designed to interfere with signaling by thismolecule will constitute a new class of antibiotics. The inventionfurther provides methods for detecting an autoinducer and methods forthe in vitro production of autoinducr-2.

I. Definitions:

Various terms relating to the biological molecules of the presentinvention are used throughout the specifications and claims. The terms“substantially the same,” “percent similarity” and “percent identity”are defined in detail below.

With reference to the novel signaling factor of the present invention,this molecule is alternatively referred to herein as “signaling factor”,“signaling molecule”, “autoinducer”, and more specifically,“autoinducer-2” or AAI-2”. The terms “autoinducer-2” and “AI-2” referspecifically to the signaling factor as produced by Vibrio harveyi. Theterms “signaling factor” or “signaling molecule”, “autoinducer” or“AI-2-like molecule” are intended to refer generally to the signalingfactors of the present invention, of which AI-2 is an example.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated nucleic acid” may comprise a DNA molecule inserted into avector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a procaryote or eucaryote. An “isolated nucleic acidmolecule” may also comprise a cDNA molecule.

With respect to RNA molecules of the invention, the term “isolatednucleic acid” primarily refers to an RNA molecule encoded by an isolatedDNA molecule as defined above. Alternatively, the term may refer to anRNA molecule that has been sufficiently separated from RNA moleculeswith which it would be associated in its natural state (i.e., in cellsor tissues), such that it exists in a “substantially pure” form (theterm “substantially pure” is defined below).

With respect to protein, the term “isolated protein” or “isolated andpurified protein” is sometimes used herein. This term refers primarilyto a protein produced by expression of an isolated nucleic acid moleculeof the invention. Alternatively, this term may refer to a protein whichhas been sufficiently separated from other proteins with which it wouldnaturally be associated, so as to exist in “substantially pure” form.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the factor of interest (e.g., pathogenesissignaling factor, nucleic acid, oligonucleotide, protein, etc.). Morepreferably, the preparation comprises at least 75% by weight, and mostpreferably 90-99% by weight, the factor of interest. Purity is measuredby methods appropriate for the factor of interest (e.g. chromatographicmethods, agarose or polyacrylamide gel electrophoresis, HPLC analysis,and the like).

With respect to antibodies of the invention, the term “immunologicallyspecific” refers to antibodies that bind to one or more epitopes of aprotein of interest, but which do not substantially recognize and bindother molecules in a sample containing a mixed population of antigenicbiological molecules.

With respect to oligonucleotides, the term “specifically hybridizing”refers to the association between two single-stranded nucleotidemolecules of sufficiently complementary sequence to permit suchhybridization under pre-determined conditions generally used in the art(sometimes termed “substantially complementary”). In particular, theterm refers to hybridization of an oligonucleotide with a substantiallycomplementary sequence contained within a single-stranded DNA or RNAmolecule of the invention, to the substantial exclusion of hybridizationof the oligonucleotide with single-stranded nucleic acids ofnon-complementary sequence.

The term “promoter region” refers to the transcriptional regulatoryregions of a gene, which may be found at the 5′ or 3′ side of the codingregion, or within the coding region, or within introns.

The term “selectable marker gene” refers to a gene encoding a productthat, when expressed, confers a selectable phenotype such as antibioticresistance on a transformed cell.

The term “reporter gene” refers to a gene that encodes a product whichis easily detectable by standard methods, either directly or indirectly.

The term “operably linked” means that the regulatory sequences necessaryfor expression of the coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toenable expression of the coding sequence. This same definition issometimes applied to the arrangement of transcription units and otherregulatory elements (e.g., enhancers or translation regulatorysequences) in an expression vector.

II. Description of the Signaling Factor

The invention provides a heterologous bio-assay that has enabled theidentification of an extracellular signaling factor produced by S.typhimurium and E. coli, among other pathogenic bacteria. The factor issometimes referred to herein as a “pathogenesis signaling” factor ormolecule, though it acts as a signal for a variety of physiologicalchanges in bacteria other than pathogenesis. The factor mimics theaction of AI-2 (autoinducer-2) of the quorum sensing bacterium Vibrioharveyi, and it acts specifically through the V. harveyi SignalingSystem 2 detector, LuxQ.

The signaling factor is a small, soluble, heat labile organic moleculethat is involved in intercellular communication in all three bacteria.In E. coli and Salmonella, maximal secretion of the molecule occurs inmid-exponential phase and the extracellular activity is degraded asglucose becomes depleted from the medium or by the onset of stationaryphase. Destruction of the signaling molecule in stationary phaseindicates that, in contrast to other quorum sensing systems, quorumsensing in bacteria that utilize the signaling molecule is critical forregulating behavior in the pre-stationary phase of growth. Proteinsynthesis is required for degradation of the activity, indicating that acomplex regulatory circuitry controls quorum sensing in these entericbacteria.

Increased signaling activity is observed if, after growing in thepresence of glucose, the bacteria are transferred to a high osmolarity(e.g., 0.4 M NaCl) or to a low pH (e.g., pH 5.0) environment. Moreover,degradation of the signal is induced by conditions of low osmolarity(e.g., 0.1 M NaCl. High osmolarity and low pH are two conditionsencountered by pathogenic enteric bacteria, such as S. typhimurium andE. coli, when they undergo the transition to a pathogenic existenceinside a host organism. Thus, quorum sensing in these bacteria appearsto play a role in regulating their virulence, by way of directing thebacteria to undergo the transition between a host-associated (i.e.,pathogenic) and a free-living existence.

Other factors that regulate the activity of the signaling molecule aredescribed in greater detail in Example 2. Particularly exemplified isthe regulation of the molecule in S. typhimurium.

The timing of lux induction in the bio-assay and the shape of theresponse curve of V. harveyi to the S. typhimurium and E. coli signalsare indistinguishable from those of V. harveyi responding to its ownSignaling System 2 inducer, AI-2. Furthermore, each of the signalingmolecules from S. typhimurium, E. coli and V. harveyi can be partiallypurified according to the same protocol. These results indicate that thesignaling molecules from each of the aforementioned organisms are eitheridentical or very closely related. Accordingly, AI-2 from V. harveyi isa signaling molecule of the invention, but appears to play a differentrole in that organism than it does in pathogenic enteric bacteria suchas Salmonella and Escherichia.

A. Structure of the AI-2 Signaling Factor

Thus, in another aspect, the invention provides autoinducer-2 (AI-2)signaling factor and derivatives thereof. AI-2 of the invention can beused to regulate bacterial growth in a variety of applications. Thepresent invention provides autoinducer-2 molecules having the structure:

wherein R₁, R₂, R₃ and R₄ are independently selected from hydrido, halo,alkyl, haloalkyl, cycloalkyl, cycloalkenyl, heterocyclyl, methyl, cyano,alkoxycarbonyl, amino, carboxyl, hydroxyl, formyl, nitro, fluoro,chloro, bromo, methyl, aryl, heteroaryl, aralkyl, heteroarylalkyl,alkylsulfonyl, haloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,hydroxyalkyl, mercaptoalkyl, alkoxyalkyl, aryloxyalkyl,heteroaryloxyalkyl, aralkyloxyalkyl, heteroarylalkyloxyalkyl,alkylthioalkyl, arylthioalkyl, heteroarylthioalkyl, aralkylthioalkyl,heteroarylalkylthioalkyl, haloalkylcarbonyl, haloalkyl(hydroxy)alkyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl,heteroarylalkylcarbonyl, carboxyalkyl, alkoxycarbonylalkyl,alkylcarbonyloxyalkyl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl,aralkylaminoalkyl, heteroarylaminoalkyl, heteroarylalkylaminoalkyl,alkoxy, and aryloxy; phenyl, cyclohexyl, cyclohexenyl, benzofuryl,benzodioxolyl, furyl, imidazolyl, thienyl, thiazolyl, pyrrolyl,oxazolyl, isoxazolyl, triazolyl, pyrimidinyl, isoquinolyl, quinolinyl,benzimidazolyl, indolyl, pyrazolyl and pyridyl, aminosulfonyl, fluoro,chloro, bromo, methylthio, methyl, ethyl, isopropyl, tert-butyl,isobutyl, pentyl, hexyl, cyano, methoxycarbonyl, ethoxycarbonyl,isopropoxycarbonyl, tert-butoxycarbonyl, propoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, pentoxycarbonyl, methylcarbonyl,fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl,difluorochloromethyl, dichlorofluoromethyl, difluoroethyl,difluoropropyl, dichloroethyl, dichloropropyl, methoxy, methylenedioxy,ethoxy, propoxy, n-butoxy, hydroxymethyl, hydroxyethyl, methoxymethyl,ethoxymethyl, trifluoromethoxy, methylamino, N,N-dimethylamino,phenylamino, ethoxycarbonylethyl, and methoxycarbonylmethyl, methyl,ethyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyano,methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyl,phenylethyl, phenylpropyl, methylsulfonyl, phenylsulfonyl,trifluoromethylsulfonyl, hydroxymethyl, hydroxyethyl, methoxymethyl,ethoxymethyl, methylcarbonyl, ethylcarbonyl, trifluoromethylcarbonyl,trifluoro(hydroxy)ethyl, phenylcarbonyl, benzylcarbonyl,methoxycarbonylmethyl, ethoxycarbonylethyl, carboxymethyl,carboxypropyl, methylcarbonyloxymethyl, phenyloxy, phenyloxymethyl,thienyl, furyl, and pyridyl, wherein the thienyl, furyl, pyridyl,methylthio, methylsulfinyl, methyl, ethyl, isopropyl, tert-butyl,isobutyl, pentyl, hexyl, cyano, fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl,dichloropropyl, methoxy, methylenedioxy, ethoxy, propoxy, n-butoxy,hydroxymethyl, hydroxyethyl and trifluoromethoxy.

The chemical groups disclosed herein are known to those of skill in theart. For example, as used herein, the term “hydrido” denotes a singlehydrogen atom (H). This hydrido radical may be attached, for example, toan oxygen atom to form a hydroxyl radical or two hydrido radicals may beattached to a carbon atom to form a methylene (—CH₂—) radical. Inaddition, alkyl radicals are “lower alkyl” radicals having one to aboutten carbon atoms. Most preferred are lower alkyl radicals having one toabout six carbon atoms. Examples of such radicals include methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,iso-amyl, hexyl and the like. The term “halo” means halogens such asfluorine, chlorine, bromine or iodine. The terms “carboxy” or “carboxyl”denotes —CO₂H. The term “carbonyl”, whether used alone or with otherterms, denotes —(S═O)—.

Preferably, the autoinducer-2 molecule of the invention is4,5-Dihidroxy-2,3-pentanedione having the structure:

As used herein, an “autoinducer-2 (AI-2)” molecule of the inventionincludes a molecule that acts as a diffusable sensor for quorum sensingSignaling System 2. For example, AI-2 can regulate gene expression byincreasing or decreasing expression of genes associated withpathogenesis of a microorganism. Typically, autoinducer molecules areproduced by microorganisms, such as bacteria, during metabolism. Forexample, the autoinducer-2 (AI-2) molecule of the invention can interactwith LuxP which is the protein encoded by the homologue of the luxP geneof pathogenic bacteria such as V. cholerae, S. typhimurium and E. coli.In turn, the AI-2-LuxP complex can interact with LuxQ which is theprotein product encoded by the luxQ gene. The AI-2-LuxP-LuxQ interactioncan promote luminescence in bacteria such as Vibrio spp. TheAI-2-LuxP-LuxQ interaction has been linked to the activation ofbiochemical pathways required for bacterial pathogenicity. Thus, theinvention provides a method for controlling bacterial gene expressionand for regulating bacterial pathogenicity by modulating AI-2-LuxP-LuxQinteractions.

In another aspect, the invention provides methods for using homocysteineas an autoinducer molecule. The structure of homocysteine is as follows:

Homocysteine is produced by the activity of the LuxS protein onS-ribosylhomocysteine (FIG. 15). Thus, the invention provides methodsfor using homoserine as an autoinducer.

The present invention also encompasses optically active isomers of anautoinducer-2 molecule. As used herein, an “isomer” is intended toinclude molecules having the same molecular formula as an autoinducer-2molecule of the invention but possessing different chemical and physicalproperties due to a different arrangement of the atoms in the molecule.Isomers include both optical isomers and structural isomers. As usedherein, “optically active” is intended to include molecules that havethe ability to rotate a plane of polarized light. An optically activeisomer includes the L-isomer and the D-isomer of an autoinducer-2molecule of the invention.

In addition to optically active isomers, analogs of an autoinducer-2molecule are included in the invention. As used herein, an AI-2 “analog”is intended to include molecules that are structurally similar but notidentical to the claimed autoinducer molecule4,5-Dihidroxy-2,3-pentanedione. Analogs of AI-2 can include moleculesthat inhibit rather than stimulate the activity of the LuxP protein. Forexample, an analog of AI-2 that is capable of a nonproductiveinteraction with LuxP can be produced. Such a molecule can retain theability to bind to LuxP, but the analog AI-2-LuxP complex will not beable to productively interact with LuxQ resulting in an inhibition ofbacterial pathogenicity. Thus, an AI-2 analog of the invention can actas an inhibitor of bacterial pathogenesis by competing with endogenousAI-2 for binding to LuxP. In addition, an analog of AI-2 can beconstructed such the analog AI-2-LuxP complex is capable ofnonproductively interacting with LuxQ. In this case, the analogAI-2-LuxP-LuxQ complex is rendered nonfunctional for subsequentbiochemical processes such as, for example, transcriptional activationof genes required for pathogenicity. The invention also includes AI-2analogs which act synergistically to enhance the ability of AI-2 toincrease the activity of the LuxP protein.

B. Preparation of the Signaling Factor

Initial strategies for purifying the signaling molecule of the inventionresulted in a partially purified preparation comprising the moleculewith a specific signaling activity estimated at about 0.1-1.0 mg of thepartially purified material stimulating a 1,000-fold increase inluminescence, as measured in the V. harveyi bioassay. The signalingactivity does not extract quantitatively into organic solvents and itdoes not bind to either a cation or an anion exchange column. Themolecule is a small (less than 1,000 kDa), polar but uncharged organicfactor. The activity is acid stable and base labile, and it is heatresistant to 80° C. but not 100° C. These features of the signalingmolecule make it clear that the molecule is different from anypreviously described autoinducer.

The signaling factor of the present invention may be purified from itsnatural sources, i.e. the bacteria that produce it. With regard topurifying AI-2 from natural sources, altering the culture medium, e.g.,by adding glucose or another sugar, by increasing the osmolarity, and/ordecreasing pH, can increase production of the signaling molecule inSalmonella and other enteric bacteria, has also enabled purification ofthe signaling molecule to near-homogeneity. Thus, the molecule has nowbeen highly purified from culture fluids of enteric bacteria (e.g., E.coli, S. typhimurium) using the following protocol:

1. Grow a culture of the signal producing enteric bacterium overnight inLB containing 0.5% glucose or another sugar (37° C., with aeration).Inoculate fresh LB containing glucose or another sugar at 0.5% with theovernight culture, at a 1:100 dilution. Grow the diluted culture tomid-exponential phase (3.5 h, 37° C., with aeration).

2. Pellet the cells (10,000 rpm, 10 min, 4° C.). Discard the culturemedium. Resuspend the cells and wash in {fraction (1/10)} th theoriginal volume of low osmolarity NaCl solution (0.1 M NaCl in water).

3. Pellet the cells again (10,000 rpm, 10 min, 4° C.). Discard the lowosmolarity culture fluid. Resuspend the cells in {fraction (1/10)} ththe original volume of high osmolarity NaCl solution (0.4 M NaCl inwater). Incubate the suspension at 37° C. for 2 h with aeration. Duringthis time, increased production and secretion of the signaling moleculeoccurs.

4. Pellet the cells (10,000 rpm, 10 min, 4° C.). Collect the supernatantcontaining the secreted signaling molecule, filter the supernatantthrough a 0.2 M bacterial filter to remove any remaining cells.

5. Evaporate the aqueous filtrate using a rotary evaporator at 30° C.Extract the dried filtrate in {fraction (1/10)} th the original volumeof chloroform:methanol (70:30).

6. Evaporate the organic extract using a rotary evaporator at roomtemperature. Re-dissolve the dried extract in methanol at {fraction(1/100)} th of the original volume.

7. Subject the partially purified signal to High Performance LiquidChromatography (HPLC), using a preparative reverse phase C18 column.Elute the molecule with a linear gradient of 0-100% acetonitrile inwater at 5 ml per minute. Collect 30 fractions, 5 ml each.

8. Assay the HPLC fractions in the V. harveyi BB170 AI-2 assay, and poolthe active fractions.

The product from the C18 column contains the signaling molecule and asmall number of other organic molecules. This highly purifiedpreparation of the signaling molecule has activity 50-100 times greaterthan that of the partially purified material described above (thepreparation of which did not include the high osmoticum step or thefinal HPLC step), i.e., 1-10 μg material stimulates a 1,000-foldincrease in luminescence in the V. harveyi bioassay.

Subsequent strategies for purifying the AI-2 signaling molecule have ledto the identification of a novel in vitro system for producing AI-2.Thus, in addition to providing a cloned, overexpressed and purified S.typhimurium LuxS protein, the present invention also provides a methodfor producing AI-2 in vitro. The present invention provides a mechanismfor generating large quantities of pure AI-2 useful for mass spectraland NMR analysis, and for screening compounds which regulate theactivity of AI-2. Moreover, the present invention provides a method fordetermining the in vivo biosynthetic pathway for AI-2 synthesis. The invitro method for AI-2 production is described below in Example 5 andFIG. 15. The method provides a novel means for efficiently producingautoinducer molecules for further study. The method also provides ameans for producing substantial quantities of AI-2 for use in commercialapplications. Such applications include, but are not limited to, addingAI-2 of the invention to a growth media to increase bacterial growth.Such a method is particularly useful in the in the production ofantibiotics from cultured bacteria. The addition of AI-2 can increasethe antibiotic production of such organisms by promoting cell growth.Preferably, the signaling factor AI-2 is produced by the in vitro methodset forth in Example 5 of the disclosure.

C. Uses of the Signaling Factor

The isolated and purified signaling molecules of the present inventionare used as targets for the design of compounds that regulate theactivity of AI-2. As used herein, “regulate” includes increasing ordecreasing the activity of AI-2. As used herein, the “activity” of AI-2encompasses any aspect of the molecules ability to act as a signalingfactor in bacterial quorum sensing. A “compound” can be any agent orcomposition that effects the activity of AI-2. For example, a compoundof the invention can be a nucleic acid, a protein or small molecule.Thus, the invention provides a means for identifying a new class ofantibiotics that inhibit the activity of the AI-2 molecule or otherwiseblock the signaling pathway in which the molecule participates. Suchinhibitors may be identified by large-scale screening of a variety oftest compounds, using the V. harveyi bioassay in the presence of thepurified signaling molecule. A reduction in signaling activity in thepresence of a test compound would be indicative of the ability of thatcompound to inhibit the activity of the signaling molecule or to blocksome other part of the pathogenesis signaling pathway.

Further, the invention provides a basis for the rational design ofspecific inhibitors or non-functional analogs of AI-2. Suchstructure-specific inhibitors or analogs may be tested in the V. harveyibioassay for their ability to inhibit the signaling molecule or to blockthe pathogenesis signaling pathway.

The invention also encompasses methods for identifying naturallyproduced compounds that inhibit the activity of a signaling moleculesuch as autoinducer-2. For example, a defensive strategy employed byeucaryotic organisms to avoid bacterial colonization is to specificallytarget and inhibit quorum sensing controlled functions. Such a mechanismhas been identified in D. pulchra. Recent studies indicate thathalogenated furanones produced by D. pulchra inhibit quorum sensing bycompeting for the homoserine-lactone (HSL) autoinducer-binding site inLuxR. Thus, by providing a novel autoinducer and the cellular componentsthat interact with the autoinducer, the present invention also providesa method to screen naturally produced compounds for their effect onquorum sensing system-2. For example, naturally produced compounds canbe screened for their effect on the autoinducer-2-LuxP interaction.Alternatively, such compounds can be screened for their effect onautoinducer-2-LuxP-LuxQ interactions.

It will be appreciated by persons skilled in the art that, now thattargets for the signaling molecule have been identified in E. coli,inhibition of the E. coli target can also be used to screen potentialsignaling molecule inhibitors or analogs. The inventors have prepared aler-lacZ reporter fusion construct to be used in testing for reductionof expression of the Type III secretion gene in E. coli O157:H7(pathogenic strain) directly. Furthermore, a similar locus exists in S.typhimurium.

Thus, the invention provides a method for selecting inhibitors orsynergists of the autoinducer-2 molecule,4,5-Dihidroxy-2,3-pentanedione. As used herein, an “inhibitor” of AI-2is intended to include molecules that interfere with the ability of theautoinducer molecule to act as a signal for luminescence orpathogenesis. Inhibitors include molecules that degrade or bind to AI-2.The method comprises contacting the autoinducer molecule with asuspected inhibitor or synergist, measuring the ability of the treatedautoinducer molecule to stimulate the activity of a selected gene thendetermining whether the suspected inhibitor or synergist represses orenhances the activity of the autoinducer molecule. Actual inhibitors andsynergists of the autoinducer molecule are then selected. For example, asuspected inhibitor can be mixed with 4,5-Dihidroxy-2,3-pentanedione andthe mixture then combined with a reporter strain of V. harveyi disclosedherein. The amount of luminescence in the presence of the suspectedinhibitor can be compared with a control mixture which does not includethe inhibitor. A decrease in luminescence is indicative of AI-2inhibition. In this manner, compounds that regulate bacterialpathogenesis can be rapidly screened.

In another aspect, the invention also provides methods of selectinginhibitory and synergistic analogs of AI-2. The method comprises mixinga known amount of the autoinducer molecule with a known amount of thesuspected inhibitory or synergistic analog, measuring the ability of thetreated autoinducer molecule to stimulate the activity of a selectedgene then determining whether the suspected inhibitory or synergisticanalog represses or enhances the activity of the autoinducer molecule.Actual inhibitory or synergistic analogs of the autoinducer molecule arethen selected.

The autoinducer-2 molecule can be purified from the native source usingconventional purification techniques, derived synthetically by chemicalmeans, or preferably, produced by the in vitro method of the inventiondescribed below. As used herein, “purified from a native source” isintended to include an autoinducer-2 molecule of the above formula thathas been manufactured by an organism. “Purified from the native source”includes isolating the autoinducer molecule from the culture media orcytoplasm of bacteria such as S. typhimurium using conventionalpurification techniques. As used herein, “synthesized by chemical means”is intended to include autoinducer molecules of the claimed formula thathave been artificially produced outside of an organism. The inventionincludes an autoinducer of the invention manufactured by a personskilled in the art from chemical precursors using standard chemicalsynthesis techniques.

The invention further provides methods of inhibiting the infectivity ofa pathogenic organism as well as therapeutic compositions containing anAI-2 analog or AI-2 inhibitor of the invention. The methods compriseadministering to a subject a therapeutically effective amount of anpharmaceutical composition that is capable of inhibiting the activity ofAI-2. As used herein, “inhibiting infectivity” includes methods ofaffecting the ability of a pathogenic organism to initially infect orfurther infect a subject that would benefit from such treatment. Apharmaceutical composition of the invention can include, but is notrestricted to, an agent that prevents the transcriptional activation ofextracellular virulence factors such as exotoxin A and elastolyticproteases. As used herein, an “agent” includes molecules that inhibitthe ability of the LuxP protein and LuxQ protein to activatetranscription of extracellular virulence factors. Agents includeinhibitors that interact directly with AI-2 such that AI-2 is preventedfrom acting as a sensor for quorum sensing Signaling System-2.Preferably, the agent interacts with 4,5-Dihidroxy-2,3-pentanedione.Agents further include analogs of AI-2 that can compete with4,5-Dihidroxy-2,3-pentanedione for binding to LuxP or LuxQ.

The invention further provides pharmaceutical compositions forpreventing or treating pathogen-associated diseases by targeting factorsinvolved in the Signaling System type-2 pathway. For example, LuxP orLuxQ, or homologues thereof, provide a common target for the developmentof a vaccine. Antibodies raised to LuxP or LuxQ, or homologues thereof,can inhibit the activation of bacterial pathways associated withvirulence. Thus, LuxP and LuxQ provide common antigenic determinantswhich can be used to immunize a subject against multiplepathogen-associated disease states. For example, the autoinducerSignaling System type-2 is believed to exist in a broad range ofbacterial species including bacterial pathogens. As discussed above, theautoinducer-2 signaling factor is believed to be involved ininter-species as well as intra-species communication. In order for thequorum sensing Signaling System type-2 to be effective for inter-speciescommunication, it is likely to be highly conserved among variousbacterial species. Thus, challenging a subject with the LuxP or LuxQpolypeptide, or an antigenic fragment thereof, isolated from aparticular organism may confer protective immunity to other diseasestates associated with a different organism. For example, a vaccinedeveloped to the LuxP protein isolated from V. cholerae may be capableof cross-reacting with a LuxP homologue expressed by a differentorganism. Thus, it is envisioned that methods of the present inventioncan be used to treat pathogen-associated disease states.

Generally, the terms “treating”, “treatment”, and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a spirochete infection or disease or sign orsymptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure for an infection or disease and/or adverse effectattributable to the infection or disease. “Treating ” as used hereincovers any treatment of (e.g., complete or partial), or prevention of,an infection or disease in a mammal, particularly a human, and includes:

(a) preventing the disease from occurring in a subject that may bepredisposed to the disease, but has not yet been diagnosed as having it;

(b) inhibiting the infection or disease, i.e., arresting itsdevelopment; or

(c) relieving or ameliorating the infection or disease, i.e., causeregression of the infection or disease.

Thus, the invention includes various pharmaceutical compositions usefulfor ameliorating symptoms attributable to a bacterial infection or,alternatively, for inducing a protective immune response to prevent suchan infection. For example, a pharmaceutical composition according to theinvention can be prepared to include an antibody against, for example,LuxP or LuxQ, a peptide or peptide derivative of LuxP or LuxQ, a LuxP orLuxQ mimetic, or a LuxP or LuxQ-binding agent according to the presentinvention into a form suitable for administration to a subject usingcarriers, excipients and additives or auxiliaries. Frequently usedcarriers or auxiliaries include magnesium carbonate, titanium dioxide,lactose, mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed.).

The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disease and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the patient. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g, in Langer, Science, 249:1527, (1990); Gilman et al. (eds.) (1990), each of which is hereinincorporated by reference.

As used herein, “administering a therapeutically effective amount” isintended to include methods of giving or applying a pharmaceuticalcomposition of the invention to a subject which allow the composition toperform its intended therapeutic function. The therapeutically effectiveamounts will vary according to factors such as the degree of infectionin a subject, the age, sex, and weight of the individual. Dosage regimacan be adjusted to provide the optimum therapeutic response. Forexample, several divided doses can be administered daily or the dose canbe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The pharmaceutical composition can be administered in a convenientmanner such as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids and othernatural conditions which may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepharmaceutical composition in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the pharmaceutical composition into a sterilevehicle which contains a basic dispersion medium and the required otheringredients from those enumerated above.

The pharmaceutical composition can be orally administered, for example,with an inert diluent or an assimilable edible carrier. Thepharmaceutical composition and other ingredients can also be enclosed ina hard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the individual's diet. For oral therapeuticadministration, the pharmaceutical composition can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationscan, of course, be varied and can conveniently be between about 5 toabout 80% of the weight of the unit. The amount of pharmaceuticalcomposition in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the pharmaceuticalcomposition can be incorporated into sustained-release preparations andformulations.

As usd herein, a “pharmaceutically acceptable carrier” is intended toinclude solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the pharmaceutical composition, usethereof in the therapeutic compositions and methods of treatment iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of pharmaceutical composition iscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the pharmaceuticalcomposition and the particular therapeutic effect to be achieve, and (b)the limitations inherent in the art of compounding such anpharmaceutical composition for the treatment of a pathogenic infectionin a subject.

The principal pharmaceutical composition is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

In addition to generating antibodies which bind to antigenic epitopes ofproteins of the invention, it is further envisioned that the method ofthe invention can be used to induce cellular responses, particularlycytotoxic T-lymphocytes (CTLs), to antigenic epitopes of, for exampleLuxP or LuxQ. Typically, unmodified soluble proteins fail to prime majorhistocompatibility complex (MHC) class I-restricted CTL responseswhereas particulate proteins are extremely immunogenic and have beenshown to prime CTL responses in vivo. CTL epitopes and helper epitopeshave been identified in proteins from many infectious pathogens.Further, these epitopes can be produced concurrently such that multipleepitopes can be delivered in a form that can prime MHC class Irestricted CTL responses. An example of a system that can producerecombinant protein particles carrying one or more epitopes entails theuse of the p1 protein of the retrotransposon Ty1 of Saccharomycescerevisiae (Adams, et al., Nature, 329:68, 1987). Sequences encoding CTLepitopes can, for example, be fused to the C-terminus of p1 and theresulting Ty virus-like particles (Ty-VLPs) may be able to generate aCTL response. Thus, conserved regions of pathogenic antigens, such asthose that are involved in, or result from, the activation of SignalingSystem type-2, can be identified and incorporated together in a particlewhich enables the host immune system to mount an effective immuneresponse against multiple spirochetal organisms. Further, the method ofthe invention can be used to generate particles with multiple epitopesto a single protein, such as LuxP, or multiple epitopes from variousproteins.

The method of the invention also includes slow release antigen deliverysystems such as microencapsulation of antigens into liposomes. Suchsystems have been used as an approach to enhance the immunogenicity ofproteins without the use of traditional adjuvants. Liposomes in theblood stream are generally taken up by the liver and spleen, and areeasily phagocytosed by macrophages. Liposomes also allow co-entrapmentof immunomodulatory molecules along with the antigens, so that suchmolecules may be delivered to the site of antigen encounter, allowingmodulation of the immune system towards protective responses.

In another embodiment, the invention provides a method for identifying acompound which binds to a protein of the invention, such as LuxP orLuxQ. The method includes incubating components comprising the compoundand LuxP or LuxQ under conditions sufficient to allow the components tointeract and measuring the binding of the compound to LuxP or LuxQ.Compounds that bind to LuxP or LuxQ include peptides, peptidomimetics,polypeptides, chemical compounds and biologic agents as described above.

Incubating includes conditions which allow contact between the testcompound and LuxP or LuxQ. Contacting includes in solution and in solidphase. The test ligand(s)/compound may optionally be a combinatoriallibrary for screening a plurality of compounds. Compounds identified inthe method of the invention can be further evaluated, detected, cloned,sequenced, and the like, either in solution or after binding to a solidsupport, by any method usually applied to the detection of a specificDNA sequence such as PCR, oligomer restriction (Saiki, et al.,Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide(ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. USA,80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, etal., Science, 241:1077, 1988), and the like. Molecular techniques forDNA analysis have been reviewed (Landegren, et al., Science,242:229-237, 1988). Also included in the screening method of theinvention are combinatorial chemistry methods for identifying chemicalcompounds that bind to LuxP or LuxQ. See, for example, Plunkett andEllman, “Combinatorial Chemistry and New Drugs”, Scientific American,April, p.69, (1997).

The invention further provides a method for promoting the production ofa bacterial product, such as, for example, an antibiotic, by contactinga culture of bacteria with an AI-2 of the invention at a concentrationeffective to stimulate or promote cellular metabolism, growth orrecovery. For example, it is known that antibiotic-producing bacteriaonly produce an antibiotic at or near the peak of log phase growth. Bycontacting a culture medium containing such antibiotic-producingbacteria with AI-2 of the invention, production of an antibiotic can beinduced at an earlier phase of growth. Thus, AI-2 of the inventionprovides a method for increasing the amount of antibiotic produced by aculture. “Culture medium”, as used herein, is intended to include asubstance on which or in which cells grow. The autoinducer molecule canbe included in commercially available cell culture media includingbroths, agar, and gelatin.

The invention further provides a method for identifying factors thatdegrade or inhibit synthesis autoinducer-2. For example, it is knownthat autoinducer-1 concentration peaks in mid-to late log phase of abacterial cell culture. In contrast, autoinducer-2 concentrationincreases earlier in log phase of bacterial cell culture growth and ispresent in lower amounts in late log phase and stationary phase. Thisdata indicates that a mechanism exists for the degradation ofautoinducer-2 at a specific point in bacterial growth. By providingisolated and purified autoinducer-2, the invention allows for theidentification of the mechanism whereby autoinducer-2 levels arecontrolled. For example, partially purified bacterial extracts can beassayed against isolated autoinducer-2 to identify those fractions whichdegrade autoinducer-2. Fractions that degrade autoinducer-2 can befurther fractionated by techniques known to those skilled in the artuntil those cellular components involved in autoinducer degradation areisolated.

The present invention also provides a method of regulating theexpression of a gene. The method comprises inserting a gene intobacteria chosen for enhancement of gene expression by an agent capableof stimulating the activity of the LuxQ protein and incubating thebacteria with an agent capable of stimulating the activity of the LuxPprotein. Thus, the signaling molecule of the invention can also be usedin screens for other targets that are regulated by the molecule. Clonedpromoter-fusion libraries can be prepared from any species of bacteriaand these libraries can be used to identify genes that are induced orrepressed by the signaling factor, simply by screening for differencesin reporter activity in petri or microtiter plates containing thesignaling molecule compared to plates that do not contain the molecule.

In addition, quorum sensing is a major regulator of biofilm control andquorum sensing blockers can therefore be used to prevent and/or inhibitbiofilm formation. Also, quorum sensing blockers are effective inremoving, or substantially decreasing the amount of, biofilms which havealready formed on a surface. Thus, by providing the structure ofautoinducer-2 (AI-2), the present invention provides a new approach toidentifying compounds which inhibit bacterial infections by regulatingbiofilm formation.

It is known that quorum sensing blockers can reduce protease productionby 50% in some strains of bacteria but the discovery that certaincompounds can substantially eliminate protease production imparts clearsignificant clinical advantages. Furthermore, the unexpected findingthat biofilm formation can be inhibited or prevented by quorum sensingblockers leads to the reasonable conclusion that other quorum sensingblockers which are known to exhibit quorum sensing blocking in othersystems, such as protease production, will also be effective againstbiofilm formation.

The compounds of the invention are advantageously used to treat and/orprevent infections, such as those caused by V. angufflarum or Aeromonasspp. Examples of this type of infection are vibriosis and furunculosisdisease in fish. Inhibition of biofilm formation by the bacteria,optionally together with a reduction or elimination of extracellularprotease production, renders the bacteria substantially non-pathogenic.The compounds of the invention may be formulated by conventional methodsfor use in the treatment and/or prevention of bacterial infection. Forexample, the compounds may be used as solid or liquid preparations (suchas tablets, suspensions or solutions for oral administration or sterileinjectable compositions), optionally together with pharmaceuticallyacceptable diluents, carriers or other additives.

For the treatment of vibriosis or furunculosis disease in fish, thecompounds or compositions containing them may be applied directly to thefish or they may be added to the fish's food or water.

In another embodiment, the invention provides a method of removing abiofilm from a surface which comprises treating the surface with acompound of the invention. The surface is preferably the inside of anaqueous liquid distribution system, such as a drinking waterdistribution system or a supply line connected to a dental air-watersystem. The removal of biofilms from this type of surface can beparticularly difficult to achieve. The compound is preferably applied tothe surface as a solution of the compound either alone or together withother materials such as conventional detergents or surfactants.

A further embodiment of the invention is an antibacterial compositioncomprising a compound of the invention together with a bacteriocidalagent. In the antibacterial compositions, the compound of the inventionhelps to remove the biofilm whilst the bacteriocidal agent kills thebacteria. The antibacterial composition is preferably in the form of asolution or suspension for spraying and/or wiping on a surface.

In yet another aspect, the invention provides an article coated and/orimpregnated with a compound of the invention in order to inhibit and/orprevent biofilm formation thereon. The article is preferably of plasticsmaterial with the compound of the invention distributed throughout thematerial.

III. Description of Nucleic Acids Encoding Proteins Involved inSignaling Factor Biosynthesis

In accordance with another aspect of the present invention, we havecloned and characterized the genes responsible for production of thesignaling molecule of the invention in V. harveyi, S. typhimurium and E.coli. These genes encode a novel family of proteins responsible forautoinducer production. We have designated the members of this family ofautoinducer production genes as luxS, specifically luxS_(E.c.),luxS_(S.t.), and luxS_(V.h.) for E. coli, S. typhimurium and V. harveyirespectively.

Mutagenesis of luxS in V. harveyi, S. typhimurium and E. coli eliminatesproduction of the signaling molecule in all three species of bacteria.S. typhimurium could be complemented to full production of the moleculeby the introduction of either the E. coli O157:H7 luxS_(E.c.) gene orthe V. harveyi BB120 luxS_(V.h.) gene. These results indicate that boththe E. coli and V. harveyi LuxS proteins can function with S.typhimurium cellular components to produce the signaling molecule. E.coli DH5 was only partially complemented to production of the signalingmolecule by the introduction of either the E. coli O157:H7 luxS_(E.c.)or the V. harveyi BB120 luxS_(V.h.) gene. Because in trans expression ofluxS genes in E. coli DH5 did not completely restore signaling moleculeproduction, other biochemical or physiological factors may contribute tosignal production.

The regulation of signaling molecule production differs betweenpathogenic and non-pathogenic strains. For example E. coli O157:H7strains produce AI-2 at 30° and 37° C. with or without glucose while E.coli K-12 strains do not produce the molecule in the absence of apreferred carbon source. And, all of the E. coli O157 strains testedproduce greater signaling activity than do non-pathogenic E. colistrains. Likewise, pathogenic S. typhimurium 14028 producessignificantly more signaling activity than does S. typhimurium LT2.

Sequence analysis shows that the LuxS proteins are highly homologous,and complementation data suggest that the proteins can function acrossspecies. These results indicate that the enzymatic activity carried outby the LuxS proteins and any other cellular machinery that contributesto synthesis of the signaling molecule are conserved. We did notidentify any amino acid sequence motif in the LuxS proteins that isindicative of a particular function. Therefore, the LuxS proteins mostlikely catalyze one specific enzymatic step in biosynthesis of thesignaling molecule. The remainder of the steps involved in signalingmolecule biosynthesis could be a consequence of normal intermediarymetabolic processes. The luxS genes identified here bear no homology toother genes known to be involved in production of acyl-homoserinelactone autoinducers (luxI-like (Fuqua et al., J. Bacteriol. 176,269-275, 1994), luxLM-ainS-like (Bassler et al, 1993, supra; Gilson etal, J. Bacteriol. 177, 6946-6951, 1995), further indicating that thesignaling molecules of the present invention are novel.

Database analysis of finished and unfinished bacterial genomes revealsthat many other species of bacteria possess a gene homologous to luxSfrom V. harveyi, S. typhimurium and E. coli. The species of bacteriaidentified and the percent homology/identity (H/I) to the LuxS proteinof V. harveyi are as follows: Haemophilus influenzae (88/72),Helicobacter pylori (62/40), Bacillus subtilis (58/38), Borreliaburgfdorferi (52/32), Neisseria meningitidis (89/80), Neisseriagonorrhoeae (89/80), Yersinia pestis (85/77), Campylobacter jejuni(85/74), Vibrio cholerae (95/90), Deinococcus radiodurans (65/45),Mycobacterium tuberculosis (59/41), Enterococcus faecalis (60/44),Streptococcus pneumoniae (57/36) and Streptococcus pyogenes (57/36). Asreported earlier (Bassler et al., 1997, supra), a few of these specieswere tested for production of the signaling molecule. We showed that V.cholerae and Y. enterocolitica but not B. subtilis produced signalingactivity. We believe that B. subtilis does produce the molecule but theenvironmental conditions that induce its synthesis have not yet beendetermined. Furthermore, we believe that all of the species identifiedin the database analysis produce an AI-2-like molecule.

The nucleotide sequences of the luxS genes from V. harveyi, E. coli andS. typhimurium are set forth at the end of the specification as SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NOS:3 and 4, respectively (the sequencesread in the 5′ to 3′ direction). These genes are sometimes referred toherein as “LuxS_(V.h.)”, “LuxS_(E.c.)” and “LuxS_(S.t.)”, respectively.The amino acid sequences deduced from SEQ ID NOS: 1-4 are set forth atthe end of the specification (and in FIG. 11) as SEQ ID NO:10, SEQ IDNO:11 and SEQ ID NO:12, respectively. It is believed that SEQ ID NOS:1and 2 constitute full-length clones, whereas SEQ ID NO:3 and SEQ ID NO:4do not.

The LuxS genes from V. harveyi, E. coli and S. typhimurium are describedin greater detail in Example 3. Although those particular LuxS genes andtheir encoded proteins are exemplified herein, this inventionencompasses LuxS genes and their encoded enzymes from any bacterialspecies, having the sequence, structural and functional properties ofthe LuxS-encoded proteins described herein. As mentioned in Example 3,homologous nucleic acid sequences have been identified in a variety ofbacterial species, but identity of those sequences as LuxS genesheretofore had not been appreciated. LuxS nucleotide and deduced aminoacid sequences from other bacterial species are set forth at the end ofthe specification as SEQ ID NOS: 5-9 and 13-17, respectively, andinclude sequences from the following species: Haemophilus influenzae,Helicobacter pylori, Bacillus subtilis, Borrelia burgdorferi and Vibriocholerae.

In addition to LuxS homologs from species other than V. harveyi, E. colior S. typhimurium, variants and natural mutants of SEQ ID NOS:1-9 arelikely to exist within different species or strains of Vibrio,Escherichia and Salmonella (indeed, E. coli strain DH5 possesses anon-functional mutant form of the gene). Because such variants areexpected to possess certain differences in nucleotide and amino acidsequence, this invention provides an isolated LuxS nucleic acid moleculeand encoded protein having at least about 50-60% (preferably 60-80%,most preferably over 80%) sequence homology in the coding region withthe nucleotide sequences set forth as SEQ ID NOS:1-9, respectively (and,preferably, specifically comprising the coding regions of SEQ IDNOS:1-9), and the amino acid sequence of SEQ ID NOS:10-17. Because ofthe natural sequence variation likely to exist among these proteins andnucleic acids encoding them, one skilled in the art would expect to findup to about 40-50% sequence variation, while still maintaining theunique properties of the LuxS-encoded proteins of the present invention.Such an expectation is due in part to the degeneracy of the geneticcode, as well as to the known evolutionary success of conservative aminoacid sequence variations, which do not appreciably alter the nature ofthe protein. Accordingly, such variants are considered substantially thesame as one another and are included within the scope of the presentinvention.

For purposes of this invention, the term “substantially the same” refersto nucleic acid or amino acid sequences having sequence variation thatdo not materially affect the nature of the protein (i.e. the structuralcharacteristics and/or biological activity of the protein). Withparticular reference to nucleic acid sequences, the term “substantiallythe same” is intended to refer to the coding region and to conservedsequences governing expression, and refers primarily to degeneratecodons encoding the same amino acid, or alternate codons encodingconservative substitute amino acids in the encoded polypeptide. Withreference to amino acid sequences, the term “substantially the same”refers generally to conservative substitutions and/or variations inregions of the polypeptide not involved in determination of structure orfunction. The terms “percent identity” and “percent similarity” are alsoused herein in comparisons among amino acid sequences. These terms areintended to be defined as they are in the UWGCG sequence analysisprogram (Devereaux et al., Nucl. Acids Res. 12: 387-397, 1984),available from the University of Wisconsin, and the parameters used bythat program are the parameters intended to be used herein to comparesequence identity and similarity.

A. Preparation of LuxS Nucleic Acid Molecules, Encoded Proteins, andImmunologically Specific Antibodies

1. Nucleic Acid Molecules

LuxS Nucleic acid molecules of the invention may be prepared by twogeneral methods: (1) They may be synthesized from appropriate nucleotidetriphosphates, or (2) they may be isolated from biological sources. Bothmethods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as the DNAshaving SEQ ID NOS:1-9, enables preparation of an isolated nucleic acidmolecule of the invention by oligonucleotide synthesis. Syntheticoligonucleotides may be prepared by the phosphoramadite method employedin the Applied Biosystems 38A DNA Synthesizer or similar devices. Theresultant construct may be purified according to methods known in theart, such as high performance liquid chromatography (HPLC). Long,double-stranded polynucleotides, such as a DNA molecule of the presentinvention, must be synthesized in stages, due to the size limitationsinherent in current oligonucleotide synthetic methods. Such longdouble-stranded molecules may be synthesized as several smaller segmentsof appropriate complementarity. Complementary segments thus produced maybe annealed such that each segment possesses appropriate cohesivetermini for attachment of an adjacent segment. Adjacent segments may beligated by annealing cohesive termini in the presence of DNA ligase toconstruct an entire 1.8 kb double-stranded molecule. A synthetic DNAmolecule so constructed may then be cloned and amplified in anappropriate vector.

LuxS Nucleic acids also may be isolated from appropriate biologicalsources using methods known in the art. In a preferred embodiment, agenomic clone is isolated from a cosmid expression library of an S.typhimurium or E. coli genome. In another embodiment, a genomic clone isisolated from a cosmid library of another bacterial genome.

In accordance with the present invention, nucleic acids having theappropriate level sequence homology with the protein coding region ofany of SEQ ID NOS:1-9 may be identified by using hybridization andwashing conditions of appropriate stringency. For example,hybridizations may be performed, according to the method of Sambrook etal., using a hybridization solution comprising: 5×SSC, 5×Denhardt'sreagent, 1.0% SDS, 100 g/ml denatured, fragmented salmon sperm DNA,0.05% sodium pyrophosphate and up to 50% formamide. Hybridization iscarried out at 37-42NC for at least six hours. Following hybridization,filters are washed as follows: (1) 5 minutes at room temperature in2×SSC and 1% SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1%SDS; (3) 30 minutes-1 hour at 37NC in 1×SSC and 1% SDS; (4) 2 hours at42-65Nin 1×SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules of a specifiedsequence homology (Sambrook et al., 1989):T _(m)=81.5C+16.6Log[Na⁺]+0.41(% G+C)−0.63(% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na⁺]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57C. The T_(m) of a DNA duplex decreases by 1-1.5Cwith every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42C.

Another way to isolate the luxS nucleic acids is to search the publiclyavailable databases for the luxS sequence in the bacterial genome ofinterest, design PCR primers from the sequence and amplify the genedirectly from the chromosome. The PCR product can then be cloned.Alternatively, if the complete sequence of a specific bacterial genomeis not available, the sequences set forth in the present invention, orany other luxS sequence, may be used to design degenerateoligonucleotides for PCR amplification and cloning of luxS from thechromosome.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell.

LuxS nucleic acid molecules of the invention include DNA, RNA, andfragments thereof which may be single- or double-stranded. Thus, thisinvention provides oligonucleotides (sense or antisense strands of DNAor RNA) having sequences capable of hybridizing with at least onesequence of a nucleic acid molecule of the present invention, such asselected segments of the DNA having SEQ ID NOS:1, 2 or 3. Sucholigonucleotides are useful as probes for detecting LuxS genes ortranscripts.

2. Proteins and Antibodies

A full-length LuxS gene product of the present invention may be preparedin a variety of ways, according to known methods. The protein may bepurified from appropriate sources, e.g., cultured bacteria such as S.typhimurium, E. coli or V. harveyi.

The availability of full-length LuxS nucleic acid molecules enablesproduction of the encoded protein using in vitro expression methodsknown in the art. According to a preferred embodiment, the enzyme may beproduced by expression in a suitable expression system. For example,part or all of a DNA molecule, such as the DNA having SEQ ID NO:1 or 2,may be inserted into a plasmid vector adapted for expression in abacterial cell, such as E. coli, or a eucaryotic cell, such asSaccharomyces cerevisiae or other yeast. Such vectors comprise theregulatory elements necessary for expression of the DNA in the hostcell, positioned in such a manner as to permit expression of the DNA inthe host cell. Such regulatory elements required for expression includepromoter sequences, transcription initiation sequences and, optionally,enhancer sequences.

The protein produced by LuxS gene expression in a recombinantprocaryotic or eucyarotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, to be easilypurified from the surrounding medium. If expression/secretion vectorsare not used, an alternative approach involves purifying the recombinantprotein by affinity separation, such as by immunological interactionwith antibodies that bind specifically to the recombinant protein. Suchmethods are commonly used by skilled practitioners.

The protein encoded by the LuxS gene of the invention, prepared by oneof the aforementioned methods, may be analyzed according to standardprocedures. For example, the protein may be subjected to amino acidsequence analysis, according to known methods. The stability andbiological activity of the enzyme may be determined according tostandard methods, such as by the ability of the protein to catalyzeproduction of the signaling molecule under different conditions.

The present invention also provides antibodies capable ofimmunospecifically binding to the LuxS-encoded protein of the invention.Polyclonal antibodies may be prepared according to standard methods. Ina preferred embodiment, monoclonal antibodies are prepared, which reactimmunospecifically with various epitopes of the protein. Monoclonalantibodies may be prepared according to general methods of Köhler andMilstein, following standard protocols. Polyclonal or monoclonalantibodies that immunospecifically interact with the LuxS-encodedproteins can be utilized for identifying and purifying such proteins.For example, antibodies may be utilized for affinity separation ofproteins with which they immunospecifically interact. Antibodies mayalso be used to immunoprecipitate proteins from a sample containing amixture of proteins and other biological molecules.

B. Uses of LuxS Nucleic Acid Molecules, Encoded Protein andImmunologically Specific Antibodies

LuxS nucleic acids may be used for a variety of purposes in accordancewith the present invention. DNA, RNA, or fragments thereof may be usedas probes to detect the presence of and/or expression of LuxS genes.Methods in which LuxS nucleic acids may be utilized as probes for suchassays include, but are not limited to: (1) in situ hybridization; (2)Southern hybridization (3) northern hybridization; and (4) assortedamplification reactions such as polymerase chain reactions (PCR).

The LuxS nucleic acids of the invention may also be utilized as probesto identify related genes from other bacteria. As is well known in theart, hybridization stringencies may be adjusted to allow hybridizationof nucleic acid probes with complementary sequences of varying degreesof homology.

As described above, LuxS nucleic acids are also used to advantage toproduce large quantities of substantially pure encoded protein, orselected portions thereof. It should be noted in this regard that thecloned genes inserted into expression vectors can be used to make largequantities of the signaling molecule itself, from any selected bacterialspecies, in a recombinant host such as E. coli DH5. Specific luxS genesare cloned, a large quantity of the encoded protein produced, therebyproducing a large quantity of the specific signaling molecule. This willbe particularly useful determining differences in the structures ofsignaling molecules from different species, if such differences arefound to exist. Alternatively, a large quantity of signaling moleculefrom the species of interest could be made using the cloned gene in anexpression vector, and thereafter used in library screens for potentialtargets in petri plate assays, as described above.

Purified LuxS gene products, or fragments thereof, may be used toproduce polyclonal or monoclonal antibodies which also may serve assensitive detection reagents for the presence and accumulation of thoseproteins in cultured cells. Recombinant techniques enable expression offusion proteins containing part or all of a selected LuxS-encodedprotein. The full length protein or fragments of the protein may be usedto advantage to generate an array of monoclonal or polyclonal antibodiesspecific for various epitopes of the protein, thereby providing evengreater sensitivity for detection of the protein in cells or tissue.Other uses of the LuxS proteins include overproduction to make aquantity of the LuxS proteins sufficient for crystallization. Solvingthe crystal structure of the LuxS proteins would enable the exactdetermination of the LuxS active site for catalysis of production of thesignaling molecule. The LuxS crystal structure can therefore be used forcomputer modeling that would greatly facilitate design of signalingmolecule analogs, LuxS inhibitors, and rational drug design in general.

Polyclonal or monoclonal antibodies immunologically specific for aLuxS-encoded protein may be used in a variety of assays designed todetect and quantitate the protein. Such assays include, but are notlimited to: (1) flow cytometric analysis; (2) immunochemicallocalization of a LuxS protein in cells or tissues; and (3) immunoblotanalysis (e.g., dot blot, Western blot) of extracts from various cellsand tissues. Additionally, as described above, antibodies can be usedfor purification of the proteins (e.g., affinity column purification,immunoprecipitation).

IV. Vibrio harveyi Screening Strain

In another aspect, the invention provides a novel strain of Vibrioharveyi having a genotype that is luxN⁻, luxS⁻. The Gram negativebacterium Vibrio harveyi contains two parallel quorum sensing circuitswhich synthesize and detect two different autoinducer molecules (FIG.13). Circuit 1 synthesizes AI-1 a HSL autoinducer similar in structureto autoinducers synthesized by the LuxI/R pathway found in other gramnegative bacteria. Circuit 2 synthesizes AI-2, the structure of whichhas not been determined. Synthesis of AI-1 and AI-2 is dependent onLuxLM and LuxS respectively. Following the buildup of a criticalexternal concentration of the autoinducers, signaling occurs via aseries of a phosphorylation/dephosphorylation reactions. The AI-1 andAI-2 detectors, LuxN and LuxQ respectively, contain both a sensor kinasedomain with a conserved histidine (H1) and an attached responseregulator domain with a conserved aspartate (D1). Signals from bothsensors are channeled to the shared integrator protein LuxU, which isphosphorylated on a histidine residue (H2). Subsequently, the signal istransduced to a conserved aspartate residue (D2) on the responseregulator protein LuxO. LuxO-phosphate controls the expression of theluciferase structural operon luxCDABE which results in the emission oflight. The presence of either AI-1 or AI-2 is sufficient to turn onlight production in wild-type V. harvyi (strain BB120). For this reason,we have V. harvyi strains containing separate mutations in Lux genes L,M, S or Q which are defective in their ability to synthesize or detectAI-1 or AI-2, respectively. AI-2 is detectable using strain BB170 whichis sensor 1⁻, sensor 2⁺ (LuxN⁻, LuxQ⁺). This strain was used to detectAI-2 in diverse bacteria. The light emission response of wild type,LuxN- and LuxQ-phenotypes to increasing cell density is shown in FIG.14.

BB170 is a sensitive reporter for AI-2, however, the BB170 strain is notoptimal for use as a reporter for inhibitors of the quorum pathway in amicrotiter based assay. The desired strain is defective in its abilityto detect AI-1 (sensor 1⁻) and defective in its ability to synthesizeAI-2. Thus, the invention provides a strain of V. harveyi that isgenotypically luxN⁻ and luxS⁻. The new strain, designated MM32, isuseful for identifying inhibitors of the quorum sensing pathway. Forexample, since the new strain is sensor 1⁻, its growth or ability toluminesce will not be affected by those organisms producing AI-1.Further, since MM32 is defective for production of AI-2, the addition ofexogenous AI-2, or analogs thereof, allows for the rapid identificationof inhibitors of AI-2.

In addition, the materials described above are ideally suited for thepreparation of a kit. Such a kit may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method.

The container means may comprise a strain of bacteria capable ofdetecting the presence of an autoinducer. Preferably, the bacterialstrain will be capable of providing an easily detectable signal in thepresence of autoinducer-2. More preferably, the desired strain isdefective in its ability to detect AI-1 (sensor 1⁻) and defective in itsability to synthesize AI-2. Thus, the kit may provide a strain of V.harveyi that is genotypically luxN⁻ and luxS⁻ designated MM32. Thebacterial strain is useful for identifying autoinducer-2 as well asinhibitors of autoinducer-2 and the quorum sensing pathway.

V. Methods for Detecting a Bacterial Biomarker

Many bacteria presently known to utilize the autoinducer-1 signalingfactor associate with higher organisms, i.e., plants and animals, atsome point during their lifecycles. For example, Pseudomonas aeruginosais an opportunistic pathogen in humans with cystic fibrosis. P.aeruginosa regulates various virulence determinants with AI. Otherexamples of AI producing bacteria include Erwinia carotovora,Pseudomonas aureofaciens, Yersinia enterocolitica, Vibrio harveyi, andAgrobacterium tumefaciens. E. carotovora infects certain plants andcreates enzymes that degrade the plant's cell walls, resulting in whatis called “soft rot disease.” Yersinia enterocolitica is a bacteriumwhich causes gastrointestinal disease in humans and has been reported toproduce an autoinducer. P. aureofaciens associates with the roots ofplants and produces antibiotics that block fungus growth in the roots.The antibiotic synthesis is under autoinducer control. The presentinvention provides novel autoinducer-2 and methods of usingautoinducer-2. In contrast to autoinducer-1, autoinducer-2 is believedto be an intra-species as well as inter-species signaling factor.Autoinducer-2 is further believed to regulate the expression ofpathogenic and virulence factors not regulated by autoinducer-1. Thus,the present invention provides a method to identify and regulate theexpression of bacterial biomarkers in, for example, pathogenic bacteria.Methods of the invention can be used to regulate the activity ofbacterial pathogens that are present in both plants and animals.

The invention further provides a method for detecting anautoinducer-associated bacterial biomarker by contacting at least onebacterial cell with an autoinducer molecule under conditions and forsuch time as to promote induction of a bacterial biomarker. As usedherein, an “autoinducer-associated bacterial biomarker” is any bacterialcell component which is regulated, modified, enhanced, inhibited orinduced in response to an autoinducer. A biomarker can be any bacterialcell component that is identifiable by known microscopial, histologicalor molecular biological techniques. Such biomarkers can be used, forexample, to distinguish pathogenic from non-pathogenic bacteria. Such abiomarker can be, for example, a molecule present on a cell surface, aprotein, a nucleic acid, a phosphorylation event or any molecular ormorphological characteristic of a bacterial cell that is modified as aresult of the bacterium being contacted with an autoinducer. Preferably,the autoinducer is autoinducer-2. The method of the invention isparticularly useful for identifying a biomarker which is indicative ofbacterial pathogenicity. As previously noted, autoinducers areextracellular signalling factors used by a variety of bacteria toregulate cellular functions in response to various environmentalstimuli, including high population density. It is believed thatpathogenic bacteria express a biomarker, such as an antigenicdeterminant, as a result of increased autoinducer concentration in thesurrounding environment. Thus, the present invention provides a methodfor identifying a biomarker by contacting a bacterium with autoinducer-2and assaying for the presence of the biomarker.

The method of the invention contemplates the use of a probe to identifya biomarker present in a bacterial cell. As used herein, a “probe” canbe a nucleic acid, protein, small molecule or antibody useful fordetecting a bacterial biomarker present in a sample. The probe can beused in a screening assay to identify a biomarker present in a sampleafter the sample has been contacted with, for example, an autoinducer.For example, a bacterial biomarker produced by a bacterium followingcontact with an autoinducer can can be identified by contacting a samplecontaining the bacterium with a probe that binds to the biomarker. Suchassays can be used to detect, prognose, diagnose, or monitor variousconditions, diseases, and disorders, or monitor the treatment thereof. Aprobe can be detectably labeled such that the probe is detectable whenbound to its target marker. Such means for detectably labeling a probeinclude a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, fluorescent, orradionuclide label. Other reporter means and labels are well known inthe art.

In addition, the method of the invention can be used to analyzedifferential gene expression in a bacterial cell following contact withan autoinducer. For example, where the expression of genes in differentcells, normally a cell of interest and a control, is compared and anydiscrepancies in expression are identified. In such assays, the presenceof discrepancies indicates a difference in the classes of genesexpressed in the cells being compared. Methods that can be used to carryout the foregoing are commonly known in the art.

The present invention provides a method for identifying a biomarkerwhich can be a protein. For example, a bacterial protein expressed inresponse to an autoinducer molecule can be detected using theappropriate antibody. The expressed protein can be, for example, anantigenic determinant indicative of a pathogenic bacterium. Antibodiesused in the method of the invention are suited for use, for example, inimmunoassays for the detection of such a determinant. The term“antibody” as used herein is meant to include intact molecules ofpolyclonal or monoclonal antibodies, as well as fragments thereof, suchas Fab and F(ab′)₂. For example, monoclonal antibodies are made fromantigen containing fragments of a protein by methods well known to thoseskilled in the art (Kohler, et al., Nature, 256:495, 1975).

In addition, the monoclonal antibodies in these immunoassays can bedetectably labeled in various ways. For example, radioisotopes may bebound to an immunoglobulin either directly or indirectly by using anintermediate functional group. Intermediate functional groups whichoften are used to bind radioisotopes which exist as metallic ions toimmunoglobulins are the bifunctional chelating agents such asdiethylenetriamine-pentacetic acid (DTPA) and ethylenediaminetetraaceticacid (EDTA) and similar molecules. Typical examples of metallic ionswhich can be bound to monoclonal antibodies are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

A probe useful in the method of the invention can also be a nucleic acidprobe. For example, nucleic acid hybridization techniques are well knownin the art and can be used to identify an RNA or DNA biomarker presentin a sample containing a bacterium contacted with an autoinducer.Screening procedures which rely on nucleic acid hybridization make itpossible to identify a biomarker from any sample, provided theappropriate probe is available. For example, oligonucleotide probes,which can correspond to a part of the sequence encoding a targetprotein, can be synthesized chemically. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. For such screening, hybridizationis preferably performed under in vitro or in in vivo conditions known tothose skilled in the art.

In addition, the materials described above are ideally suited for thepreparation of a kit. Such a kit may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. A kitof the invention may contain a first container means comprising isolatedautoinducer-2. The isolated autoinducer-2 can be used to regulate theexpression of a biomarker in a target bacterium. For example,autoinducer-2 can be used to induce expression of a particular biomarkerwhich can then be identified by a probe. Thus, the kit may contain asecond container means comprising a probe that can be detectablylabeled. The kit may also have a third container comprising areporter-means, such as a biotin-binding protein, such as avidin orstreptavidin, bound to a reporter molecule, such as an enzymatic,fluorescent, or radionuclide label. Other reporter means and labels arewell known in the art. For example, the kit of the invention may providereagents necessary to perform nucleic acid hybridization analysis asdescribed herein or reagents necessary to detect antibody binding to atarget.

The following description sets forth the general procedures involved inpracticing this aspect of the present invention. To the extent thatspecific materials are mentioned, it is merely for purposes ofillustration and is not intended to limit the invention. Unlessotherwise specified, general cloning procedures, such as those set forthin Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory(1989) (hereinafter “Sambrook et al.”) or Ausubel et al. (eds) CurrentProtocols in Molecular Biology, John Wiley & Sons (1998) (hereinafter“Ausubel et al.”) are used.

EXAMPLE 1 Quorum Sensing in Escherichia coli and Salmonella typhimurium

There have been preliminary indications that E. coli senses cell density(Huisman et al., Science 265: 537-539, 1994; Sitnikov et al., Proc.Natl. Acad. Sci. USA 93: 336-341, 1996; Garcia-Lara et al., J.Bacteriol. 178: 2742-2748, 1996). We took advantage of the reducedselectivity of the Signaling System 2 sensor in V. harveyi to develop asensitive assay for detection of extracellular signal molecules producedby E. coli and S. typhimurium. Using this assay we could determine theconditions under which many strains of E. coli and S. typhimuriumsynthesize, secrete, and degrade a signaling substance that willinteract with the V. harveyi System 2 detector.

Materials and Methods

Preparation of cell-free culture fluids. E. coli strains AB1157 and DH5and S. typhimurium strain LT2 were grown at 30° C. overnight withaeration in LB broth containing glucose at the concentrations specifiedin the text. The following morning fresh LB medium containing the sameconcentration of glucose used for the overnight growth was inoculated ata 1:100 dilution with the overnight grown cultures. The fresh cultureswere grown for various times at 30° C. with aeration. Cell-free culturefluids were prepared by removing the cells from the growth medium bycentrifugation at 15,000 rpm for 5 min in a microcentrifuge. The clearedculture fluids were passed through 0.2 m HT Tuffryn filters (Gelman) andstored at −20° C. Cell-free culture fluids containing V. harveyiAutoinducer-2 were prepared from V. harveyi strain BB152 (Autoinducer1⁻, Autoinducer 2⁺). V. harveyi BB120 (Autoinducer 1⁺, Autoinducer 2⁺)was used to prepare culture fluids containing Autoinducer-1. In bothcases, the V. harveyi strains were grown overnight at 30° C. withaeration in AB (Autoinducer Bioassay) (Bassler et al., 1993, supra)medium. Cell-free culture fluids from V. harveyi were prepared from theovernight culture exactly as described above for E. coli and S.typhimurium.

Assay for production of signaling molecules. Cell-free culture fluidsfrom E. coli, S. typhimurium and V. harveyi strains were tested for thepresence of signaling substances that could induce luminescence in theV. harveyi reporter strain BB170 or BB886. In the assays, 10 l ofcell-free culture fluids from E. coli AB1157, E. coli DH5, and S.typhimurium LT2 strains grown and harvested as described above wereadded to 96-well microtiter dishes. The V. harveyi reporter strain BB170or BB886 was grown for 16 h at 30° C. with aeration in AB medium,diluted 1:5000 into fresh AB medium, and 90 l of the diluted cells wereadded to the wells containing the E. coli and S. typhimurium cell-freeculture fluids. Positive control wells contained 10 l of cell-freeculture fluid from strain V. harveyi BB152 (Autoinducer-1⁻,Autoinducer-2⁺) or V. harveyi BB120 (Autoinducer-1⁺, Autoinducer-2⁺).Negative control wells contained 10 l of sterile growth medium. Themicrotiter dishes were shaken in a rotary shaker at 175 rpm at 30° C.Every hour, light production was measured using a Wallac Model 1450Microbeta Plus liquid scintillation counter in the chemiluminescencemode. The V. harveyi cell density was measured by diluting the samealiquots of cells used for measuring luminescence, spreading thedilutions onto solid LM medium (Bassler et al., 1993, supra), incubatingthe plates overnight at 30C, and counting the resulting colonies thefollowing day.

Preparation of E. coli and S. typhimurium viable and UV-killed cells forthe activity assay. E. coli AB1157, E. coli DH5 and S. typhimurium LT2cultures were grown for 8 h in LB containing 0.5% glucose at 30° C. withaeration. The cultures were subjected to centrifugation for 5 min at15,000 rpm in a microcentrifuge and the growth medium was removed fromthe cell pellets by aspiration. The cell pellets were resuspended in ABmedium and washed by vigorous mixing. The cells were again subjected tocentrifugation for 5 min at 15,000 rpm. The AB wash medium was removedand discarded and the cells were resuspended in fresh AB medium. Eachcell suspension was diluted to give 1×10⁶ cells/10 l, and multiple 10 laliquots were added to wells of microtiter dishes. Half of the cellaliquots were treated with short wavelength ultraviolet light for 15 minat a distance of 10 cm. This treatment was sufficient to kill all of thecells as judged by plating and incubating the UV-treated cells, andensuring that no growth occurred by the next day. 90 l of the diluted V.harveyi reporter strain BB170 was next added to the wells containingeither the viable or dead E. coli and S. typhimurium cells, and theactivity assay was carried out exactly as described in the previoussection.

Analysis of glucose in S. typhimurium LT2 culture fluids. Glucoseconcentrations were determined in cell-free culture fluids prepared fromS. typhimurium using a Trinder assay (Diagnostic Chemicals Ltd.)according to the recommendations of the manufacturer, except that theglucose standards were prepared in LB medium. The assay was sensitive toless than 0.002% glucose. No interfering substances were present in LBmedium or spent LB culture fluids.

Results and Discussion

E. coli AB1157 and S. typhimurium LT2 produce a signaling substance thatspecifically induces one of the two quorum sensing systems of V.harveyi. The V. harveyi reporter strain BB170 has the quorum sensingphenotype Sensor 1⁻, Sensor 2⁺. It induces lux expression in response toextracellular signals that act exclusively through the Signaling System2 detector. Addition of 10% cell-free spent culture fluid prepared fromV. harveyi strain BB152 (which contains the System 2 autoinducer)stimulates the reporter strain roughly 1000-fold over the endogenouslevel of luminescence expression. In FIG. 1, the light production by V.harveyi BB170 induced by the addition of 10% cell-free spent culturefluids is normalized to 100% activity.

E. coli strain AB1157 and S. typhimurium strain LT2 were grown for 8 hin LB broth or LB broth containing 0.5% glucose. The E. coli and S.typhimurium cells were removed from the growth medium and the cell-freeculture fluids were prepared and assayed for an activity that couldinduce luminescence expression in V. harveyi. Addition of 10% cell-freeculture fluid from S. typhimurium LT2 or E. coli AB1157 grown in LBcontaining glucose maximally induced luminescence in the reporter strainBB170, similar to culture fluids from V. harveyi BB152 (FIG. 1A).Specifically, E. coli AB1157 produced 106% and S. typhimurium produced237% of the V. harveyi BB152 activity. When the E. coli and S.typhimurium were grown in LB without added glucose they did not producethe signaling factor. Substitution of 10% (v/v) of LB medium containing0.5% glucose did not stimulate luminescence in the reporter strain,indicating that there is no substance in the LB-glucose growth mediumthat induces luminescence expression in V. harveyi. We tested obviouscandidates for the signal including glucose, amino acids, cAMP, acetate,homoserine lactone, -ketoglutarate and other keto acids that are knownto be excreted. None of these compounds has activity. These resultssuggest that V. harveyi BB170 can respond to some substance secreted byE. coli AB1157 and S. typhimurium LT2 when they are grown on LBcontaining glucose.

Analogous experiments were performed with the V. harveyi reporter strainBB886 (Sensor 1⁺, Sensor 2⁻). V. harveyi BB886 is defective in itsresponse to signaling molecules that act through the Signaling System 2detector, but it is an otherwise wild type strain (Bassler et al., Mol.Microbiol. 13: 273-286, 1994). FIG. 1B shows the normalized 100%activation of V. harveyi BB886 by cell-free spent culture fluidsprepared from V. harveyi BB120. V. harveyi BB120 produces the System 1autoinducer N-(3-hydroxybutanoyl)-L-homoserine lactone (Bassler et al.,1993, supra). Addition of S. typhimurium LT2 and E. coli AB1157cell-free culture fluids to V. harveyi strain BB886 caused a 5% and a 1%increase above the control level (FIG. 1B). Together the results ofFIGS. 1A and 1B show that the signaling molecule produced by E. coli andS. typhimurium must act specifically through V. harveyi Signaling System2 and not some other, unidentified pathway.

Viable E. coi AB1157 and S. typhimurium LT2 are required for secretionof the signaling molecule. We considered the possibility that growth ofE. coli AB1157 and S. typhimurium LT2 in LB medium containing glucosesimply allowed them to utilize and therefore remove some pre-existinginhibitor of induction of luminescence. To show that the cellsthemselves produce the soluble signaling factor, we added washed E. coliand S. typhimurium cells directly to the luminescence assay. Theseresults are presented in FIG. 2. In this experiment, E. coli AB1157 andS. typhimurium LT2 were grown for 8 h in LB containing 0.5% glucose; theconditions for maximal production of the signaling factor. The cellswere removed from the LB-glucose growth medium by centrifugation, andsterile V. harveyi luminescence assay medium was used to wash andresuspend the cell pellets. 1×10⁶ E. coli AB1157 or S. typhimurium LT2cells were added to the diluted V. harveyi BB170 culture at the start ofthe experiment. In FIG. 2, the left-hand bar in each series shows thatthe presence of washed E. coli AB1157 or S. typhimurium LT2 cells issufficient to fully induce luminescence in V. harveyi BB170. E. coliAB1157 and S. typhimurium LT2 stimulated lux expression in V. harveyiBB170 821-fold and 766-fold respectively. Identical aliquots of thewashed E. coli or S. typhimurium cells were killed with short waveultraviolet light prior to addition to the assay. When dead cells wereincluded in the assay, no stimulation of luminescence occurred. In FIG.2, these results are shown in the right-hand bar for each strain. Takentogether, the results show that the stimulatory factor is produced bythe E. coli AB1157 and S. typhimurium LT2 cells themselves during thetime course of the experiment; the factor could not have come from themedium in which the cells had been grown. This factor is activelyreleased into the medium by E. coli and S. typhimurium because deadcells have no activity.

E. coli DH5 does not produce the signaling activity. Clinical isolatesof E. coli and Salmonella also produce the signaling compound. Tenclinical isolates of Salmonella and five pathogenic isolates of E. coliO157 were assayed and all produced the activity. It was conceivable thatthe signal was some normal byproduct of glucose metabolism that simplydiffuses out of the cells. This is not the case however, because we showthat E. coli DH5, which is equally capable of utilizing glucose as E.coli AB1157 and S. typhimurium LT2, does not produce the signalingactivity. FIG. 1A demonstrates that unlike E. coli AB1157 and S.typhimurium LT2, the addition of 10% cell-free culture fluid preparedfrom E. coli DH5 grown 8 h in LB containing 0.5% glucose does notstimulate light production in V. harveyi BB170. Similarly, inclusion ofwashed viable or killed E. coli DH5 cells in the luminescence assay doesnot stimulate V. harveyi BB170 to produce light (FIG. 2). The inabilityof E. coli DH5 to produce the activity indicates that this highlydomesticated strain lacks the gene or genes necessary for either theproduction or the export of the signaling activity. We assayed otherlaboratory strains of E. coli for the signaling activity (Table 1). OnlyE. coli DH5 was completely defective in producing the extracellularsignal.

TABLE 1 The induction of luminescence in V. harveyi reporter strainBB170 by cell-free culture fluids from V. harveyi, S. typhimurium and E.coli is shown. Cell-free culture fluids were prepared from variousstrains of V. harveyi, S. typhimurium and E. coli as described andtested for production of a signaling substance that could stimulatelight production in the reporter strain V. harveyi BB170. The level ofV. harveyi stimulation was normalized to 100%. The data for the 5 h timepoint are shown. Species and Strain Induction of luminescence (%) V.harveyi 100 V. harveyi BB152 Salmonella 237 S. typhimurium LT2 E. coliE. coli AB1157 106 E. coli DH5 5 E. coli JM109 76 E. coli MG1655 100 E.coli MC4100 93

Glucose regulates the production and degradation of the signaling factorby S. typhimurium LT2. Cell-free culture fluids from S. typhimurium LT2and E. coli AB1157 cells grown in LB without added glucose did notstimulate the expression of luminescence in the reporter strain,indicating that metabolism of glucose is necessary for the production ofthe signal. We tested other carbohydrates, and in general, growth in thepresence of PTS sugars (see Postma et al., in Escherichia coli andSalmonella Cellular and Molecular Biology, (F. C. Niehardt, ed), Am.Soc. Microbiol., Washington D.C., pp. 1149-1174, 1996) enabled E. coliAB1157 and S. typhimurium LT2 to produce the signal. Of the sugarstested, growth on glucose induced the synthesis of the highest level ofactivity. Growth on other carbon sources, for example TCA cycleintermediates and glycerol, did not induce significant production of thesignaling activity.

We tested whether the presence of glucose was required for the cells tocontinue to produce the signal. FIG. 3 shows results with S. typhimuriumLT2 grown in LB containing limiting (0.1%) and non-limiting (0.5%)glucose concentrations. FIG. 3A shows that when glucose is limiting, S.typhimurium LT2 produces the signal in mid-exponential phase (after 4 hgrowth), but stops producing the signaling activity once glucose isdepleted from the medium. FIG. 3B shows that when glucose does notbecome limiting, S. typhimurium LT2 produces greater total activity andcontinues to produce the signaling activity throughout exponentialphase, with maximal activity at 6 h growth. Furthermore, the Figure alsoshows that the signaling activity synthesized by mid-exponential phasecells is degraded by the time the cells reach stationary phase. Inconditions of limiting glucose, no activity remained at stationaryphase, and when glucose was plentiful, only 24% of the activityremained. Increasing the concentration of glucose in the growth mediumdid not change these results, i.e., the activity was secreted duringmid-exponential growth and severely reduced activity remained in thespent culture fluids by stationary phase.

In sum, the results presented in this example show that E. coli and S.typhimurium produce a signaling substance that stimulates one specificquorum sensing system in V. harveyi. Many other bacteria have previouslybeen assayed for such an activity, and only rarely were speciesidentified that are positive for production of this factor (Bassler etal., 1997, supra). Furthermore, as shown here, the E. coli and S.typhimurium signal is potent, these bacteria make activity equal to thatof V. harveyi. The degradation of the E. coli and S. typhimurium signalprior to stationary phase indicates that quorum sensing in thesebacteria is tuned to low cell densities, suggesting that quorum sensingin E. coli and S. typhimurium is modulated so that the response to thesignal does not persist into stationary phase. Additionally, quorumsensing in E. coli and S. typhimurium is influenced by severalenvironmental factors. The production and the degradation of the signalare sensitive not only to growth phase but also to the metabolicactivity of the cells. These results indicate that the quorum sensingsignal in E. coli and S. typhimurium has two functions; it allows thecells to communicate to one another their growth phase and also themetabolic potential of the environment.

Understanding the regulation of quorum sensing in E. coli and S.typhimurium is important for understanding community structure andcell-cell interactions in pathogenesis. In the wild, pathogenic E. coliand S. typhimurium may never reach stationary phase because dispersionis critical. It is therefore appropriate that quorum sensing in E. coliand S. typhimurium should be functioning at low cell density. Thissituation is in contrast to that of V. fischeri, the luminescent marinesymbiont, where the quorum sensing system is only operational at highcell densities; cell densities indicative of existence inside thespecialized light organ of the host. The specific quorum sensing systemsof V. fischeri and E. coli and S. typhimurium appear appropriatelyregulated for the niche in which each organism exists. In both cases,quorum sensing could be useful for communicating that the bacteriareside in the host, not free-living in the environment. Additionalcomplexity exists in the E. coli and S. typhimurium systems becausethese bacteria channel both cell density information and metabolic cuesinto the quorum sensing circuit. Again, signals relaying informationregarding the abundance of glucose or other metabolites couldcommunicate to the bacteria that they should undergo the transition froma free-living mode to the mode of existence inside the host.

Under all the conditions we have tested, the signaling activitydescribed in this example does not extract quantitatively into organicsolvents and it does not bind to either a cation or anion exchangecolumn. Preliminary characterization indicates that the signal is asmall (less than 1000 MW) polar but apparently uncharged organiccompound. The activity is acid stabile and base labile, it is heatresistant to 80 but not 100° C. Purification of the E. coli, S.typhimurium and V. harveyi signal is described in greater detail in thefollowing examples.

EXAMPLE 2 Regulation of Autoinducer Production in Salmonella typhimurium

In this example, the conditions under which S. typhimurium LT2 producesAI-2, the extracellular factor that stimulates lux expression in the V.harveyi Sensor 1⁻, Sensor 2⁺ reporter strain, are elucidated. Productionof the signaling molecule by S. typhimurium occurs during growth onpreferred carbohydrates that, upon utilization by the bacteria, resultin a decrease in the pH of the medium. Lowering the pH of the growthmedium in the absence of a preferred carbon source induces limitedproduction of the factor, indicating that the cells are influenced byboth the changing pH and the utilization of the carbon source. Thesignaling activity is degraded by the time the cells reach stationaryphase, and protein synthesis is required for degradation of theactivity. Osmotic shock following growth on an appropriate carbon sourcegreatly increases the amount of activity present in the S. typhimuriumculture fluids. This increased activity is apparently due to inductionof synthesis of the autoinducer and repression of degradation of theactivity. E. coli and S. typhimurium possess a protein called SdiA whichis homologous to LuxR from V. fischeri (Wang et al., EMBO J. 10:3363-3372, 1991; Ahmer et al., J. Bacteriol. 180: 1185-1193, 1998). SdiAis proposed to respond to an extracellular factor (Sitnikov et al.,Proc. Natl. Acad. Sci. USA 93: 336-341, 1996; Garcia-Lara et al, J.Bacteriol. 178: 2742-2748, 1996), and it has been shown to controlvirulence factor production in S. typhimurium (Ahmer et al., 1998,supra). The analysis set forth below shows that the AI-2 autoinducersignaling activity does not function through the SdiA pathway.

Materials and Methods

Strains and Media. The bacterial strains used in this study and theirgenotypes and phenotypes are listed in Table 2.

TABLE 2 Bacterial strains; their genotypes and relevant phenotypes.Strain Genotype Relevant Phenotype S. typhimurium LT2 Wild type E. coliO157 Wild type E. coli MG1655 F⁻, ilvG, rfb-50 Wild type E. coli MC4100(lac)U169, araD139, rpsL, thi LacZ⁻ E. coli DH5 supE44, hsdR17, recA1,AI-2⁻ endA1, gyrA96, thi-1, relA1 V. harveyi BB170 luxN::Tn5 Sensor 1⁻,Sensor 2⁺ V. harveyi BB152 luxL::Tn5 AI-1⁻, AI-2⁺ V. harveyi JAF305luxN::Cm^(r) Sensor 1⁻, Sensor 2⁺

Luria broth (LB) contained 10 g Bacto Tryptone (Difco), 5 g YeastExtract (Difco) and 10 g NaCl per liter (Sambrook et al., 1989). Therecipe for Autoinducer Bioassay (AB) medium has been reported previously(Greenberg et al., Arch. Microbiol. 120: 87-91, 1979). LM medium(L-Marine) contains 20 g NaCl, 10 g Bacto Tryptone, 5 g Bacto YeastExtract and 15 g Agar per liter (Bassler et al., 1994, supra).Regulation of AI-2 production similar to that reported here was alsoobserved with the ATCC strain Salmonella enterica Serovar Typhimurium14028, an independent clinical isolate of Salmonella enterica SerovarTyphimurium, and nine other Salmonella enterica serovars (other thanTyphimurium).

Growth conditions for S. typhimurium LT2 and preparation of cell-freeculture fluids. S. typhimurium LT2 was grown overnight in LB broth withshaking at 30° C. The next day, 30 l of the overnight culture was usedto inoculate 3 ml of fresh LB broth. In cultures containing additionalcarbon sources, at the time of inoculation, 20% sterile stock solutionswere added to give the specified final concentrations. Followingsubculturing of the cells, the tubes were shaken at 200 rpm at 30C forthe time periods indicated in the text. Cell-free culture fluids wereprepared by removal of the cells from the culture medium bycentrifugation for 5 min at 15,000 rpm in a microcentrifuge. The clearedsupernatants were passed through 0.2 m cellulose acetate Spin X filters(CoStar, Cambridge, Mass.) by centrifugation for 1 min at 8000×g.Samples were stored at −20C. Similar results to those reported here wereobtained when we grew the S. typhimurium at 37° C. The preparation ofcell-free culture fluids from V. harveyi strains has already beenreported (Bassler et al, 1993, supra; Bassler et al., 1997, supra).

Density-dependent bioluminescence assay. The V. harveyi reporter strainBB170 (Sensor 1⁻, Sensor 2⁺) (Bassler et al., 1993, supra) was grown for12 h at 30° C. in AB medium, and diluted 1:5000 into fresh AB medium.Luminescence was measured as a function of cell density by quantitatinglight production at different times during growth with a Wallac Model1409 liquid scintillation counter (Wallac Inc., Gaithersburg, Md.). Thecell density was measured by diluting the same aliquots of cells usedfor measuring luminescence, spreading the dilutions onto solid LMmedium, incubating the plates overnight at 30° C., and counting theresulting colonies the following day. Relative Light Units are (countsmin⁻¹ ml⁻¹×10³)/(colony forming units ml⁻¹). Cell-free culturesupernatants from V. harveyi or S. typhimurium strains were added to afinal concentration of 10% (v/v) at the time of the first measurement.In control experiments, 10% (v/v) of AB medium, LB medium or LB mediumcontaining 0.5% glucose was added instead of cell-free culture fluids.

S. typhimurium autoinducer activity assay. The quorum sensing signalingactivity released by S. typhimurium LT2 was assayed following growthunder various conditions. 10 l of cell-free culture fluids from S.typhimurium LT2 grown and harvested as described above were added to96-well microtiter dishes. The V. harveyi reporter strain BB170 wasgrown overnight and diluted as described above. 90 l of the diluted V.harveyi cells were added to the wells containing the S. typhimuriumcell-free culture fluids. Positive control wells contained 10 l ofcell-free culture fluid from V. harveyi BB152 (AI-1⁻, AI-2⁺) (Bassler etal., 1993, supra). The microtiter dishes were shaken in a rotary shakerat 200 rpm at 30° C. Light production was measured hourly using a WallacModel 1450 Microbeta Plus liquid scintillation counter designed formicrotiter dishes (Wallac Inc., Gaithersburg, Md.). In theseexperiments, the cell density was not measured at each time point.Rather, to ensure that increased light production was due to a signalingactivity and not a growth medium component, the luminescence productionby V. harveyi in wells containing cell-free culture fluids was comparedto that produced by V. harveyi in wells containing 10 l of the identicalgrowth medium alone. Data are reported as fold-stimulation over thatobtained for growth medium alone.

Factors controlling signal production in S. typhimurium. S. typhimuriumLT2 was grown for 6 h in LB containing 0.5% glucose as described above.The mid-exponential phase culture was divided into several identicalaliquots. One aliquot of cells was grown to stationary phase (24 h at30° C. with shaking). In the remaining aliquots, the cells were removedfrom the LB-glucose growth medium by centrifugation for 5 min at 15,000rpm in a microcentrifuge. The resulting cell pellets were resuspended atan OD₆₀₀ of 2.0 in either LB, LB+0.5% glucose, LB at pH 5.0, or in 0.1 MNaCl, or 0.4 M NaCl (in water). The resuspended cells were shaken at 30°C. or 43° C. for 2 h. Cell-free fluids were prepared from the stationaryphase culture, and from the cells that had been resuspended andincubated in the various media or the osmotic shock solutions. Thecell-free fluids were tested for signaling activity in the S.typhimurium activity assay as described above.

Effects of growth phase, pH, glucose concentration and osmolarity onautoinducer production by S. typhimurium. S. typhimurium LT2 was grownat 30° C. for various times in LB containing limiting (0.1%) andnon-limiting (1.0%) glucose concentrations. At the times specified inthe text, the cell number was determined by plating dilutions of the S.typhimurium cultures onto LB medium and counting colonies the followingday. The pH of the two cultures was measured, and the percent glucoseremaining in each culture was determined using the Trinder assay asdescribed in Example 1. Cell-free culture fluids were prepared from theLB-glucose cultures as described above. The same cells from which thecell-free culture fluids were prepared were resuspended in 0.4 M NaClosmotic shock solution and shaken at 200 rpm, 30° C. for 2 h. Wedetermined that this timing was optimal for production of autoinducer.The cells were removed from the osmotic shock solution by centrifugationat 15,000 rpm for 5 min in a microcentrifuge. Cell-free osmotic shockfluids were prepared from the resuspended cells exactly as described forcell-free culture fluids. Signaling activity in both the cell-freeculture fluids and the cell-free osmotic shock fluids was assayed asdescribed above. In experiments in which the pH was maintained at 7.2,the cells were grown in LB+0.5% glucose containing 50 mM MOPS at pH 7.2.The pH was adjusted every 15-30 min using 1 M MOPS pH 7.2. Inexperiments performed at pH 5.0, LB broth was maintained between pH 5.0and 5.2 with 1M NaOH.

Requirement for protein synthesis in signal production, release anddegradation by S. typhimurium LT2. S. typhimurium LT2 was pre-grown inLB containing 0.5% glucose at 30° C. to an OD₆₀₀ of 2.5 (approximately6-8 h). The culture was divided into four identical aliquots. Twoaliquots were treated with 100 g/ml Cm for 5 min at room temperatureafter which the cells were harvested by centrifugation at 15,000 rpm for5 min. One Cm-treated cell pellet was resuspended in 0.1 M NaClcontaining 30 g/ml Cm, and the second pellet was resuspended in 0.4 MNaCl containing 30 g/ml Cm. Each of these pellets was resuspended to afinal OD₆₀₀ of 2.0. The remaining two culture aliquots were not treatedwith Cm. Instead, the cells in these two aliquots were harvested bycentrifugation and resuspended in 0.1 M and 0.4 M NaCl exactly asdescribed for the Cm-treated cells. The cell suspensions were incubatedat 30° C. with shaking. At the times indicated in the text, 1.5 mlaliquots were removed from the cell suspensions and cell-free osmoticshock fluids were prepared by the procedure described above.

Analysis of the effect of autoinducer on SdiA regulated gene expression.A sequence that includes the ftsQ1p and ftsQ2p promoters (Wang et al.,1991, supra) was amplified from E. coli MG1655 chromosomal DNA using thefollowing primers:

-   -   ftsQ1p, 5′-CGGAGATCTGCGTTTCAATGGATAAACTACG-3′ (SEQ ID NO: 19);    -   ftsQ2p, 5′-CGCGGATCCTCTTCTTCGCTGTTTCGCGTG-3′ (SEQ ID NO: 20).        The amplified product contained both the ftsQ promoters and the        first 14 codons of the ftsQ gene flanked by BamHI and BglII        sites. The ftsQ1p2p PCR product was cloned into the BamHI site        of vector pMLB1034 (Silhavy et al., Experiments with Gene        Fusions, Cold Spring Harbor Press, 1984) to generate a lacZ        fusion that contained the promoters, ribosome binding site, and        initiation codon of ftsQ. A correctly oriented clone, pMS207,        and a clone containing the ftsQ1p2p insert in the opposite        orientation, pMS209, were chosen for further analysis. Both        inserts were sequenced to ensure that no errors were introduced        during the PCR reaction.

For ftsQ regulation in E. coli, the plasmids pMS207 and pMS209 weretransformed into E. coli strain MC4100 (Silhavy et al., 1984, supra),and the transformants were grown overnight in LB containing 100 mg/Lampicillin at 30° C. with aeration. For rck regulation, S. typhimuriumstrains BA1105 (rck::MudJ) and BA1305 (rck::MudJ sdiA) were grownovernight in LB containing 100 mg/L kanamycin at 30° C. with aeration.The overnight cultures were diluted 20-fold into fresh medium and grownfor an additional 4.5 h. At this time, each culture was divided intofive identical aliquots and 10% (v/v) of one of the following was addedto each aliquot: LB, 0.4 M NaCl, 0.4 M osmotic shock fluids from S.typhimurium LT2, E. coli O157 or E. coli strain DH5 (negative control).The osmotic shock fluids were prepared as described above, followingpre-growth of the S. typhimurium LT2 and E. coli in LB containing 0.5%glucose for 6h. The cell suspensions were incubated at 30° C. for 2 h,after which standard -galactosidase reactions were performed on thesamples (Miller, A Short Course in Bacterial Genetics, Cold SpringHarbor Laboratory Press, 1992).

Results

S. typhimurium LT2 produces an autoinducer-like activity. In Example 1it was demonstrated that S. typhimurium and E. coli strains produce asignaling activity that stimulates lux expression in V. harveyi, and thesignaling molecule acts exclusively through the V. harveyi quorumsensing System 2. FIG. 4 shows the induction of luminescence in the V.harveyi System 2 reporter strain BB170 (Sensor 1⁻, Sensor 2⁺). Thecharacteristic quorum sensing behavior of V. harveyi is shown in thecontrol experiment (closed circles). Immediately after dilution intofresh medium, the light emitted per cell by V. harveyi drops rapidly,over 1000-fold. At a critical cell density, which corresponds to theaccumulation of a critical concentration of endogenously producedautoinducer (AI-2) in the medium, the luminescence per cell increasesexponentially, approximately 3 orders of magnitude, to again reach thepre-dilution level.

Addition of 10% cell-free culture fluid prepared from V. harveyi BB152(AI-1⁻, AI-2⁺) caused the reporter strain to maintain a high level oflight output following dilution (open circles). The increased lightoutput is due to the V. harveyi BB170 cells responding to the presenceof AI-2 in the cell-free culture fluids prepared from V. harveyi strainBB152 (Bassler et al., 1993, supra). Similarly, addition of cell-freeculture fluid from S. typhimurium LT2 grown in LB+0.5% glucose inducedluminescence in the reporter strain approximately 800-fold over thecontrol level (solid squares). No activity similar to V. harveyi AI-1was produced by S. typhimurium LT2 under these conditions and there isno AI-1 or AI-2 activity in LB+0.5% glucose (see Example 1).

Environmental factors influence autoinducer production and degradationin S. typhimurium. Control of autoinducer production in S. typhimuriumis different than in other described quorum sensing systems. FIG. 5Ademonstrates three important aspects of the regulation of autoinducerproduction in S. typhimurium. First, no autoinducer activity is observedwhen S. typhimurium is grown for 6 h in LB in the absence of glucose.Second, growth in the presence of glucose for 6 h results in substantialproduction of autoinducer (760-fold activation of the reporter strain).Third, activity, while detectable, is severely reduced when the S.typhimurium culture is allowed to grow to stationary phase (33-foldactivation of the reporter strain).

We subjected S. typhimurium LT2 to several different treatmentsincluding some environmental stresses in order to begin to understandwhat conditions favor autoinducer production versus those that favorautoinducer degradation. In the experiment presented in FIG. 5B, the S.typhimurium cells were induced for signal production by pre-growth in LBcontaining 0.5% glucose for 6 h. We have shown that under theseconditions, the glucose is not depleted (Surette and Bassler, 1998).After the induction phase of growth, the culture fluid was removed andaliquots of the cells were resuspended and incubated for 2 h under avariety of conditions that are described in the description of FIG. 2.Following each of these treatments cell-free fluids were prepared andtested for activity on BB170.

It is important to note that in the results presented in FIG. 5B, the S.typhimurium were pre-induced for autoinducer production at the start ofthe experiment, i.e., their cell-free culture fluid activated thereporter strain 760-fold. FIG. 5B shows that removal of the pre-growthculture fluid from these cells and resuspension of the cells in LBwithout glucose, in 0.1 M NaCl (hypotonic conditions), or heat shock at43° C. for 2 h resulted in no or very low autoinducer production. Theseresults indicate that the above treatments result in termination ofautoinducer production, or degradation of newly released autoinducer, orboth.

In contrast to the above results, resuspension of pre-induced cells infresh LB+glucose resulted in continued high-level production ofautoinducer (735-fold activation of the reporter). Similarly, acidic pHpromoted continued production of autoinducer (600-fold activation), andhypertonic osmotic shock (0.4 M NaCl) resulted in 1300-fold induction ofthe reporter. Increased AI-2 activity was only observed in the pH 5.0fluids or 0.4 M NaCl osmotic shock fluids of cells that were alreadyactively producing AI-2, i.e., if glucose was not included during thepre-growth, no measurable activity was produced following the identical2 h treatments.

Shifting S. typhimurium cells from LB+glucose to 0.4 M NaCl resulted inan accumulation of AI-2 activity to a level much greater than thatobserved under any other condition tested. Below it is shown that S.typhimurium cells resuspended in 0.4 M NaCl increase the biosynthesisand/or release of autoinducer, and furthermore they apparently do notdegrade significant quantities of the released activity. A similarincrease in AI-2 production occurs when the S. typhimurium cells areresuspended in 0.4 M NaCl, 0.4 M KCl or 0.8M sucrose, indicating thatthe NaCl effect on AI-2 production is an osmotic one, not an ionic one.This apparent osmotic shock effect on the S. typhimurium cells wasextremely useful because it enabled us to measure maximal release ofautoinducer activity in the absence of loss due to degradation.

The effect of glucose on signal production in S. typhimurium. In Example1 we showed that the continued presence of glucose was required for S.typhimurium to produce the quorum sensing signaling factor. Becausesugar utilization both increases the growth rate while decreasing the pHof the culture, we further analyzed the effect of metabolism of glucose,decreasing pH and increasing cell number on signal production by S.typhimurium. In the experiment presented in FIG. 6, we measured signalproduction, growth rate, and pH in growing S. typhimurium LT2 culturescontaining limiting (0.1%) and non-limiting (1.0%) concentrations ofglucose. In the data presented in FIG. 6, at various times, the level ofautoinducer produced in both the cell-free culture fluids and in thecorresponding 0.4 M NaCl osmotic shock fluids was measured andnormalized for 1×10⁹ cells. It should be noted that unlike in FIG. 5,the cells in this experiment were not pre-induced for signal production.

FIG. 6 shows that the pattern of production and disappearance ofautoinducer observed in 0.4 M NaCl osmotic shock fluids mimics thatobserved in cell-free culture fluids. However, at every time point thatautoinducer is produced, much greater activity is detected in theosmotic shock fluids than in the corresponding cell-free culture fluids.Under conditions of limiting (0.1%) glucose (FIGS. 6A, 6C and 6E), S.typhimurium produces the signaling activity between 2-4 h (Bars).However, the glucose becomes completely depleted at 4 h, and at thattime production of the factor ceases (FIG. 6A). In contrast, when thecells are grown in 1.0% glucose (FIGS. 6B, 6D, and 6F), glucose ispresent in the medium throughout the entire experiment (FIG. 6B). Underthese conditions, the cells continue to synthesize activity for 12hours. Similar to the results shown in FIG. 5 and those reported inExample 1, almost no activity was observed in cell-free culture fluidsor osmotic shock fluids from stationary phase cells at 24 h regardlessof the glucose concentration.

S. typhimurium grows at roughly the same rate in both high and lowglucose media during exponential phase. In fact, the S. typhimuriumculture grown in high glucose medium does not reach the cell densityachieved by the S. typhimurium grown in the low glucose medium (FIGS. 6Cand 6D). Cell growth is probably inhibited in this culture by thedramatically reduced pH that occurs from increased glucose utilization.These results show that the higher level of activity produced by S.typhimurium in the LB containing 1% glucose is not due to higher cellnumber, but due to induction of signal production caused by glucosemetabolism.

FIGS. 6E and 6F show the pH of the low and high glucose cultures at eachtime point. Under conditions of low glucose (FIG. 6E), the pH of theculture initially decreases as the cells utilize the glucose. However,simultaneous to the complete depletion of the glucose, the pH begins torise. In contrast, under conditions of high glucose, the pH of themedium decreases to below pH 5 (FIG. 6F). In the experiments presentedin FIG. 6, both glucose catabolism and decreasing pH occursimultaneously suggesting that either or both of these factors could beresponsible for signal production by S. typhimurium.

Both glucose metabolism and low pH independently control signalproduction in S. typhimurium. To distinguish between the contributionfrom glucose metabolism and that from low pH in signal production by S.typhimurium, we compared the activity produced by S. typhimurium grownin LB containing 0.5% glucose in a culture in which the pH wasmaintained at 7.2 (FIG. 7A), to that produced by S. typhimurium grown inLB without glucose where the pH was maintained at 5.0 (FIG. 7B). Again,we measured the signal present in cell-free culture fluids and in 0.4 MNaCl osmotic shock fluids. Similar to the data presented in FIG. 3, thelevel of signal observed in cell-free culture fluids was lower than thatobserved in the 0.4 M osmotic shock fluids.

When S. typhimurium was grown in LB+0.5% glucose at pH 7.2 increasingamounts of the quorum sensing signal were detected for 6 h. At 6 h, in0.4 M NaCl osmotic shock fluids, there was an approximately 550-foldstimulation of light production of the V. harveyi reporter strain BB170.No activity was produced after the 6 h time point. FIG. 7A shows thatthe pH was maintained between 7.15 and 7.25 for 8 h, after this time,the pH of the culture no longer decreased, but began increasingpresumably because the cells had depleted the glucose. We allowed the pHto continue to increase for the duration of the experiment. Also shownin the Figure is the cell number at each time point. At pH 7.2, thecells grew rapidly and reached a high cell density.

Analysis of time courses similar to those presented here, has shown thatS. typhimurium does not produce any signal when it is grown in LBwithout glucose at neutral pH (see Example 1). However, S. typhimuriumdid transiently produce the quorum sensing factor in the absence ofglucose when grown at pH 5.0 (FIG. 7B). Signal was produced for 4 h, andabout 450-fold stimulation of the reporter was the maximum activityachieved in 0.4 M NaCl osmotic shock fluids. Very little signal wasproduced by 5 h, and signal was completely absent after 6 h ofincubation. FIG. 7B shows that the pH was maintained between 5.0 and 5.2in this experiment. Note that the cells grew much more slowly at pH 5.0than at pH 7.2.

Preliminary characterization of the S. typhimurium autoinducerdegradative apparatus. The quorum sensing activity produced by S.typhimurium LT2 is degraded by the onset of stationary phase. We havedetermined that the activity contained in cell-free culture supernatantsand 0.4 M NaCl osmotic shock fluids from cells grown for 6 h inLB+glucose is stable for at least 24 h at 30° C., indicating that nodegradative activity is present in these cell-free fluids. Furthermore,mixing cell-free culture fluids prepared from actively producing S.typhimurium (i.e., from cultures grown for 6 h in LB+glucose) withcell-free culture fluids prepared from S. typhimurium that have degradedthe factor (i.e., from cultures grown for 12 or 24 h in LB+glucose) doesnot result in degradation of the activity. This result indicates thatthe degradative activity is not released, but instead, is associatedwith the cells.

We show in FIG. 5 that no further autoinducer is produced if S.typhimurium cells that are actively releasing autoinducer are shifted to0.1 M NaCl. However, when these same cells are shifted to 0.4 M NaCl, weobserve even greater autoinducer production. This result implies thatlow osmolarity could be a signal that induces the autoinducerdegradative machinery. To begin to analyze the mechanism by whichosmolarity affects autoinducer production and degradation in S.typhimurium, we investigated the requirement for protein synthesis insignal production and degradation by S. typhimurium in high and lowosmolarity. As described in the legend to FIG. 5, S. typhimurium LT2 wasgrown in LB containing 0.5% glucose to achieve maximal induction ofsignal production then treated with 0.1 M or 0.4 M NaCl in the presenceand absence of protein synthesis. Cell-free fluids were prepared andtested for signaling activity. Because half of the cell-free osmoticshock fluids contained chloramphenicol (Cm), V. harveyi JAF305 was usedas the reporter strain in the activity assay. This V. harveyi straincontains a Cm^(r) cassette in the luxN gene, and its phenotype is Sensor1⁻, Sensor 2⁺, a phenotype identical to that of V. harveyi BB170.

When the cells were resuspended in 0.4 M NaCl, the S. typhimuriumproduced and released increasing amounts of the signal for 200 min (FIG.8A, open squares). After this time, the level of signaling activitypresent in the cell-free osmotic shock fluid decreased somewhat,suggesting that some of the released signal was degraded. Quitedifferent results were obtained when the S. typhimurium cells wereresuspended in 0.1 M NaCl (FIG. 8B, open squares). In this case, atearly time points, the S. typhimurium produced a quantity of activityequivalent to that produced by cells resuspended in 0.4 M NaCl. However,by 120 min, no activity remained in the cell-free low osmolarity fluid.This result indicates that under conditions of low osmolarity, thereleased activity is rapidly degraded. We do not observe degradation ofthe activity in cell-free culture fluids, indicating that thedisappearance of the activity from low osmolarity cell-free fluids isnot due to chemical instability of the signaling molecule.

Under conditions of high osmolarity, when the cells were treated with Cmto inhibit protein synthesis, only about one quarter of the activity wasproduced compared to untreated cells. The closed squares in FIG. 8A showthat 300-fold induction of the reporter strain occurred in the presenceof Cm as compared to 1200-fold induction with the untreated cells (FIG.8A, open squares). When the S. typhimurium was resuspended in lowosmolarity (FIG. 8B), roughly three-quarters of the activity produced inthe absence of Cm (open squares) was produced in the presence of Cm(closed squares). In the presence of Cm, the released activity was notdegraded by 300 min in high osmolarity and only partially degraded inlow osmolarity.

To show that high osmolarity does not inhibit AI-2 signal degradation,we added the activity contained in the 0.4 M NaCl cell-free osmoticshock fluids to S. typhimurium cells that had been resuspended in 0.1 MNaCl for two hours. As shown in FIG. 8, these are cells that can degradethe factor. Table 3 shows that these S. typhimurium cells degradedgreater than 98% of the signaling activity while incubated at highosmolarity. The table also shows that S. typhimurium cells that had beenincubated in 0.4 M NaCl (these are cells that are actively producing thesignal) released no further activity when resuspended in the 0.1 M NaClincubation fluid obtained from the actively degrading cells.Furthermore, mixing active and inactive 0.4 M and 0.1 M cell-freeosmotic fluids did not result in degradation of the activity in the 0.4M fluids.

TABLE 2 High osmolarity induces release and low osmolarity inducesdegradation of the S. Typhimurium signaling factor. TreatmentFold-induction of luminescence 0.1 M NaCl activity^(a) 4 0.4 M NaClactivity^(a) 944 0.1 M cells + 0.4 M activity^(b) 17 0.4 M cells + 0.1 Mactivity^(c) 6 ^(a) S. typhimurium was grown for 6 h in LB containing0.5% glucose. The cells were pelleted and resuspended in either 0.1 M or0.4 M NaCl for 2 h. Cell-free fluids were prepared and tested foractivity. ^(b) S. typhimurium cells that had been incubated in 0.1 MNaCl for two hours were pelleted and resuspended in the activitycontained in the cleared osmotic shock fluids obtained from cellssuspended in 0.4 M NaCl for 2 h. Cell-free fluids were prepared after a2 h incubation and assayed for signaling activity. ^(c) S. typhimuriumcells that had been suspended in 0.4 M NaCl were pelleted and incubatedfor 2 h in the cleared osmotic shock fluids obtained from cellssuspended for 2 h in 0.1 M NaCl. Cell-free fluids were prepared afterthe 2 h incubation and assayed for signaling activity.

The LuxR homolog SdiA is not involved in response to the AI-2autoinducer. A gene homologous to luxR of V. fischeri has beenidentified in E. coli and S. typhimurium and is called sdiA. Two reportssuggest that in E. coli, SdiA modestly regulates the expression of thecell division locus ftsQAZ in response to a factor present in cell-freeculture fluids (Garcia-Lara et al., 1996, supra), and in response to afew homoserine lactone autoinducers (Sitnikov, et al., 1996, supra).Completion of the sequence of the E. coli genome shows that no LuxIhomologue exists in E. coli so the locus responsible for thebiosynthesis of the hypothesized soluble factor(s) has not beendetermined. Overexpression of SdiA in S. typhimurium has recently beenshown to influence the expression of several ORFs located on the S.typhimurium virulence plasmid (Ahmer, et al., 1998, supra). As in the E.coli studies, SdiA activity in S. typhimurium is proposed to bemodulated by an extracellular factor.

It was possible that the AI-2 autoinducer that we have beencharacterizing in S. typhimurium and E. coli acted through SdiA. Wetested whether AI-2 had an effect on genes regulated by SdiA in E. coliand S. typhimurium. In E. coli, we assayed an ftsQ1p2p-lacZ reporter,and in S. typhimurium we assayed an rck::MudJ fusion in both an sdiA⁺and sdiA⁻ background. We tested the effects of addition of LB, 0.4 MNaCl, 0.4 M NaCl osmotic shock fluids containing AI-2 activity from S.typhimurium LT2, E. coli O157, and 0.4 M NaCl osmotic shock fluid fromE. coli DH5. We have shown previously in Example 1 that DH5 does notproduce AI-2 activity under our growth conditions.

For the E. coli experiments we determined that MC4100 and MC4100/pMS209(containing ftsQ1p2p in the incorrect orientation) had no measurable-galactosidase activity. The level of -galactosidase produced byMC4100/pMS207 (containing the ftsQ1p2p-lacZ fusion) was roughly 20-30Miller units, and this level of activity did not change under any of theconditions tested here. This level of activity of the fusion wascomparable to that reported previously (Sitnikov et al., 1996, supra;Garcia-Lara et al., 1996, supra). In the S. typhimurium SdiA studies,similar to Ahmer et al. (1998, supra), we obtained ˜30 Miller units ofrck::MudJ activity in the sdiA⁺ background and this level was reduced to10 units in the sdiA⁻ background. No change in -galactosidase productionoccurred following the addition of AI-2 from E. coli or S. typhimurium.These results indicate that, if an extracellular factor exists thatmodulates the activity of SdiA, under the conditions we have tested, itis not AI-2.

Discussion

Quorum Sensing in E. coli and S. typhimurium. We have developed aheterologous bio-assay that enables us to detect an extracellularsignaling factor produced by S. typhimurium. The factor mimics theaction of AI-2 of the quorum sensing bacterium V. harveyi, and it actsspecifically through the V. harveyi Signaling System 2 detector LuxQ.Results using lacZ fusions to the ftsQ and rck promoters indicate that,under our assay conditions, the AI-2 quorum sensing factor does notsignal to SdiA, at least with respect to regulation of these genes. TheAI-2 quorum sensing system is therefore involved in a different S.typhimurium and E. coli signal transduction pathway than the one(s)investigated previously.

S. typhimurium LT2 produces an amount of activity roughly equivalent tothat produced by V. harveyi. We observe approximately 800-foldstimulation of the V. harveyi reporter strain BB170 upon addition of 10%S. typhimurium cell-free culture fluids. The timing of lux induction andthe shape of the response curve of V. harveyi to the S. typhimuriumsignal are indistinguishable from those of V. harveyi responding to itsown AI-2. Furthermore, we have been successful at partially purifyingboth the V. harveyi AI-2 and the S. typhimurium signal molecule usingidentical purification procedures. These two results lead us to believethat the S. typhimurium signaling molecule is identical to or veryclosely related to AI-2 of V. harveyi.

Growth Conditions Regulate Signal Production and Degradation in S.typhimurium. In this example, we further characterize the regulation ofthe signaling activity in S. typhimurium LT2. Accumulation of signalingactivity in S. typhimurium culture supernatants is maximal duringmid-exponential phase when the cells are actively utilizing glucose inrich medium. Under these growth conditions, utilization of glucose isaccompanied by a rapid drop in pH of the culture. The resultsdemonstrate that either glucose metabolism or low pH is sufficient toinduce S. typhimurium LT2 to produce the quorum sensing factor,indicating that both glucose and acidity generate independent signalsfor autoinducer production. In the presence of glucose, when the pH isnot maintained, probably both the decreasing pH and the presence of anappropriate carbon source contribute to the regulation of quorum sensingin S. typhimurium. The results also show that production of theautoinducer ceases prior to stationary phase in the presence of glucoseat neutral pH and in the absence of glucose at low pH. Therefore, acombination of acidic conditions and the absence of glucose is notrequired to cue S. typhimurium to terminate production of autoinducer.

In addition to glucose, growth on several other carbohydrates alsoinduces production of the signaling activity. These include both PTS(fructose, mannose, glucitol, and glucosamine) and non-PTS (galactoseand arabinose) sugars. These findings eliminate an exclusive role forthe PTS in the regulation of autoinducer biosynthesis. When the S.typhimurium LT2 are grown on several other carbon sources (acetate,glycerol, citrate and serine) no significant accumulation of signalingactivity is observed. We have demonstrated in Example 1 that the signalis not any of a number of substances known to be secreted by S.typhimurium including the major products of mixed acid fermentation.Clearly, production of the signaling molecule is precisely regulated bythe cells and is favored under conditions of growth on preferredcarbohydrates for reasons that we do not yet understand. Identificationof the signaling molecule and cloning of the biosynthetic gene(s) willaid in a fuller understanding of the regulation process.

Results presented in this example show that, in contrast to other quorumsensing systems, the S. typhimurium signal does not accumulate instationary phase. At least two competing processes contribute to thisregulation; autoinducer production and autoinducer degradation. In thisexample we are defining autoinducer production as an increase in thesignaling activity present in cell-free fluids. We recognize that anincrease in activity could result from release of newly biosynthesizedautoinducer, release of stored autoinducer, repression of degradation ofautoinducer, or some combination of these activities. We defineautoinducer degradation as the disappearance of signaling activity fromthe cell-free fluids. This disappearance could be due to destruction ofthe autoinducer, re-uptake of the autoinducer, or a combination of theseactivities. It is possible that under some of the conditions used in ourstudies, autoinducer production and autoinducer degradation areoccurring simultaneously. If this is the case, the activity detected incell-free culture fluids is a measure of which of these processes,production or degradation, predominates. These findings indicate thatquorum sensing in S. typhimurium is regulated such that the signal andpresumably the response to the signal do not persist into stationaryphase. Because the utilization of a preferred carbohydrate is alsorequired for signal production, quorum sensing in S. typhimurium may beused both for measuring the cell density and for measuring the potentialof the environment for growth.

Osmolarity Influences Signal Production and Degradation in S.typhimurium. S. typhimurium cells that are actively producing signal canbe further stimulated to produce signal by specific environmentaltreatments, indicating that several independent regulatory pathwayschannel information into autoinducer synthesis. One of these treatmentsis 0.4 M NaCl osmotic shock. When autoinducer producing S. typhimuriumcells are resuspended in 0.4 M NaCl, the cells release significantlygreater activity when they have the capability to synthesize proteinthan when protein synthesis is blocked. Furthermore, degradation of thesignal also requires protein synthesis. These results have severalimplications. First, in the presence of Cm, S. typhimurium resuspendedat both high and low osmolarity produce a similar amount of activity.This result indicates that, following growth in the presence of glucose,the S. typhimurium cells have a pre-defined capacity to producesignaling activity (and/or to release already synthesized activity fromthe cell). Second, when the cells are resuspended at high osmolarity,signal production increases well beyond this level. This increase insignal production requires protein synthesis, and we interpret this tomean that high osmolarity is one environmental cue that induces S.typhimurium to synthesize more of the biosynthetic apparatus necessaryfor signal production and/or release. Third, under conditions of lowosmolarity, we observe an initial release of activity, followed by arapid degradation of the activity. And, signal degradation requiresprotein synthesis because it is not observed in the presence of Cm.These results imply that the environment has changed from conditionsfavoring autoinducer production (LB+a preferred carbohydrate, or highosmolarity) to conditions where autoinducer production is not favored(low osmolarity, or absence of a preferred carbon source). Thisenvironmental change induces S. typhimurium to synthesize the protein(s)required for degradation of the signaling activity.

When the S. typhimurium cells were incubated in 0.4 M NaCl nosignificant degradation of the activity occurred by 200 min. This resultindicates that either the necessary degradative protein(s) are notsynthesized under these conditions, or alternatively, the degradativeapparatus is assembled, but its activity is inhibited by highosmolarity. The results show that high osmolarity does not inhibitsignal degradation, because cells induced to degrade the activity can doso at high osmolarity. Therefore, the persistence of the activity in thehigh NaCl samples occurs because the degradation machinery is notsynthesized, not because its activity is inhibited.

It is difficult to precisely determine when S. typhimurium cells areautoinducer producers and when they are autoinducer degraders becauseboth processes could occur simultaneously. It appears, however, that noor very low degradation occurs in high osmolarity, and conversion ofcells from overall signal producers to overall signal degraders occursin low osmolarity and requires protein synthesis. Our preliminarycharacterization of the degradative process indicates that it iscell-associated because the autoinducer activity is stable in cell-freeculture supernatants for long periods of time, and combining active withinactive cell-free culture fluids or active and inactive high and lowosmolarity cell-free fluids does not promote degradation of theautoinducer. We have recently isolated a S. typhimurium mutant that doesnot produce the AI-2 activity. If this mutant retains the capability todegrade autoinducer, analysis of it will be informative forunderstanding the timing of degradation, and for identifying the cuesthat induce the degradative machinery. We are currently attempting toisolate S. typhimurium mutants capable of autoinducer production butincapable of autoinducer degradation.

The Role for Quorum Sensing in Salmonella Pathogenesis. The observationspresented here on the regulation of signal production and degradation byS. typhimurium LT2 implicate a role for quorum sensing in pathogenesisof Salmonella. The conditions favoring signal production (nutrient rich,high osmolarity and low pH) are those likely to be encountered upon thefirst interaction of an enteric pathogen with its host. Conditionsfavoring degradation of the signal (nutrient poor, low osmolarity) arethose most probably encountered as the pathogen exits the host. Theinitial colonization of the host may be a concerted effort between apopulation of cells coordinated through this cell-cell signaling system.Other cues, that we have not yet tested, could also regulate quorumsensing in S. typhimurium. These may represent independent oroverlapping signaling pathways involved in pathogenesis. We areisolating S. typhimurium mutants to test these hypotheses. Finally,Salmonella pathogenesis is a dynamic process of interaction between thehost and metabolically active bacteria. Consistent with a role forquorum sensing in pathogenesis, our evidence suggests that this quorumsensing system is not functioning during stationary phase. We have shownthat the signaling molecule is not produced during stationary phase, andfurthermore, existing signal is degraded. Perhaps quorum sensing iscritical for S. typhimurium to undergo the transition between ahost-associated and a free-living existence.

EXAMPLE 3 Quorum Sensing in Escherichia coli, Salmonella typhimurium andVibrio harveyi: A New Family of Genes Responsible for AutoinducerProduction

In this example we report the analysis of a gene responsible for AI-2production in V. harveyi, E. coli and S. typhimurium. The geneidentified in all three species of bacteria is highly homologous, and wepropose that these genes define a new family of proteins involved inautoinducer production. The genes, which we named luxS_(V.h.),luxS_(E.c.), and luxS_(S.t.) have been identified in many species ofbacteria by genome sequencing projects, but until now no function hasbeen ascribed to this gene in any organism. The luxS genes do not bearhomology to any other gene known to be involved in autoinducerproduction.

Materials and Methods

Bacterial strains, media and recombinant DNA techniques. V. harveyiBB120 is the wild type strain (Bassler et al., 1997, supra). S.typhimurium strain LT2 was obtained from Dr. K. Hughes (University ofWashington), S. typhimurium 14028 is ATCC strain 14028 Organism:Salmonella choleraesuis. E. coli O157:H7 is a clinical isolate suppliedby Dr. Paddy Gibb (University of Calgary). Luria broth (LB) contained 10g Bacto Tryptone (Difco), 5 g Yeast Extract (Difco) and 10 g NaCl perliter. The recipe for Autoinducer Bioassay (AB) medium has been reportedpreviously (Greenberg, E. P., Hastings, J. W., and Ulitzur, S. (1979)Arch. Microbiol. 120, 87-91). Where specified, glucose was added from asterile 20% stock to a final concentration of 0.5%. Antibiotics wereused at the following concentrations (mg/L): Ampicillin (Amp) 100,Chloramphenicol (Cm) 10, Gentamycin (Gn) 100, Kanamycin (Kn) 100, andTetracycline (Tet) 10. DNA isolation, restriction analysis andtransformation of E. coli was performed as described by Sambrook et al.Probes for Southern Blot analysis were labeled using the Multiprime DNAlabeling system of Amersham. Sequencing was carried out using an AppliedBiosystems sequencing apparatus. The V. harveyi BB120 genomic librarywas constructed in the cosmid pLAFR2 as described (Bassler et al, 1993,supra). The method for Tn5 mutagenesis of cloned V. harveyi genes, andthe allelic replacement technique for inserting Tn5 mutated genes intothe V. harveyi chromosome have been reported (Bassler et al., 1993,supra).

Bioluminescence Assay. The AI-2 bioassay using the V. harveyi reporterstrain BB170 (Sensor 1⁻, Sensor 2⁺) has been discussed in the previousexamples. Cell-free culture fluids from V. harveyi, E. coli, or S.typhimurium strains to be tested for AI-2 activity were prepared asdescribed above, and assayed at 10% (v/v). AI-2 activity is reported asthe fold-induction of the reporter strain over background, or as thepercent of the activity obtained from V. harveyi BB120 (wild type)cell-free culture fluid.

Mutagenesis and analysis of the AI-2 production gene in S. typhimuriumLT2. MudJ insertion mutants of S. typhimurium LT2 were generated using aphage P22 delivery system as described (Maloy, S. R., Stewart, V. J.,and Taylor, R. K. (1996) Genetic analysis of pathogenic bacteria: alaboratory manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Following growth to mid-exponential phase in LBcontaining 0.5% glucose, the S. typhimurium insertion mutants weretested for AI-2 production using the V. harveyi BB170 bioassay. The siteof the MudJ insertion that inactivated the AI-2 production function inS. tyhimurium was identified by PCR amplification and sequencing of thechromosomal DNA at the insertion junction. A two-step amplificationprocedure was used (Caetano-Annoles, G. (1993) Meth. Appl. 3, 85-92). Inthe first PCR reaction, the arbitrary primer5′-GGCCACGCGTCGACTAGTACNNNNNNACGCCC-3′ (SEQ ID NO: 21), and the MudJspecific primer 5′-GCACTACAGGCTTGCAAGCCC-3′ (SEQ ID NO: 22) were used.Next, 1 l of this PCR reaction was used as the template in a second PCRamplification employing a second arbitrary primer(5′-GGCCACGCGTCGACTAGTCA-3′)(SEQ ID NO: 23) and another MudJ specificprimer (5′-TCTAATCCATCAGATCCCG-3′) (SEQ ID NO: 24). The PCT product fromthe second reaction was purified and sequenced.

Cloning and sequencing of the E. coli MG1655, E. coli O157:H7, and E.coli DH5 AI-2 production genes. The DNA sequence obtained from the S.typhimurium LT2 MudJ screen was used to search the E. coli MG1655 genomesequence to identify the corresponding E. coli region (Blattner et al.,Science 277, 1453-1462, 1997). The gene identified from the sequencingproject had the designation ygaG. Primers that flanked the ygaG gene andincorporated restriction sites were designed and used to amplify the E.coli MG1655, E. coli 0157:H7 and E. coli DH5 ygaG genes. The primersused are: 5′-GTGAAGCTTGTTTACTGACTAGATC-3′ (SEQ ID NO: 25) and5′-GTGTCTAGAAAAACACGCCTGACAG-3′ (SEQ ID NO: 26). The PCR products werepurified, digested and cloned into pUC19. In each case, the PCR productsfrom three independent reactions were cloned and sequenced.

Results

Identification and cloning of the gene responsible for AI-2 productionin V. harveyi. We have discussed in previous examples that, unlike manyother E. coli strains, E. coli strain DH5 does not produce an AI-2signal molecule that can be detected by V. harveyi. We reasonedtherefore, that we could use E. coli DH5 as a mutant to clone the V.harveyi AI-2 production gene. A library of wild type V. harveyi BB120genomic DNA was transformed into E. coli strain DH5, and thetransformants were screened for AI-2 production in the V. harveyi BB170AI-2 detection bioassay. The library consisted of 2,500 clones eachcontaining roughly 25 kb of V. harveyi genomic DNA. Five DH5 clones wereidentified that resulted in upwards of 300-fold stimulation of thereporter strain in the bioassay.

The recombinant cosmid DNA from the five AI-2 producing E. coli DH5clones was analyzed by restriction analysis and Southern blotting. Allfive of the cosmids contained an overlapping subset of identical V.harveyi genomic restriction fragments, indicating that we had cloned thesame locus several times. One cosmid, called pBB2929 was selected forfurther analysis. Random mutagenesis using transposon Tn5 was carriedout on cosmid pBB2929, and pools of cosmids harboring Tn5 insertionswere subsequently transformed into E. coli DH5. We tested 962 individualE. coli DH5/pBB2929::Tn5 strains for the loss of the ability to produceAI-2. Four E. coli DH5 strains harboring Tn5 insertions in pBB2929 wereidentified that failed to produce AI-2. We mapped the locations of theseTn5 insertions in pBB2929 and found that all four transposon insertionsresided in the same 2.6 kb HindIII V. harveyi genomic DNA fragment (FIG.9A).

Cosmid pBB2929 was digested with HindIII and the 8 resulting fragmentswere subcloned in both orientations into pALTER (Promega). The pALTERsubclones were transformed into E. coli DH5, and subsequently tested forAI-2 production. The only strains capable of producing AI-2 containedthe 2.6 kb HindIII fragment identified in the Tn5 mutagenesis. Thisfragment was sequenced, and only one open reading frame (ORF) could beidentified, and its location corresponded to the map positions of thefour Tn5 insertions that eliminated AI-2 production. We named the ORFluxS_(V.h.) (FIG. 9A).

Mutagenesis of luxS_(V.h.) in V. harveyi. We analyzed the effects ofluxS_(V.h.) null mutations on AI-2 production in V. harveyi. The fourTn5 insertions that mapped to the luxS_(V.h.) gene and the control Tn5insertion adjacent to the luxS_(V.h.) locus were transferred to thecorresponding locations in the V. harveyi BB120 chromosome to makestrains MM37, MM30, MM36, MM38 and MM28 respectively (FIG. 9A). Southernblotting was used to confirm the correct placement of all five Tn5insertions in the V. harveyi chromosome. The four V. harveyiluxS_(V.h.)::Tn5 insertion strains were tested for the ability toproduce AI-2, and all four strains gave identical results.

In FIG. 10A, we show the AI-2 production phenotypes of the wild typecontrol Tn5 insertion strain MM28 and one representativeluxS_(V.h.)::Tn5 insertion strain, MM30. V. harveyi MM28 and MM30 weregrown to high cell density, after which cell-free culture fluids wereprepared. The culture fluids were assayed for AI-2 activity by theability to induce luminescence in the AI-2 detector strain BB170. FIG.10A shows that addition of culture fluids from the control Tn5insertionstrain MM28 induced luminescence in the reporter 780-fold, while culturefluid from the luxS_(V.h.)::Tn5 insertion strain MM30 did not induce theexpression of luminescence in the reporter. Therefore, a null mutationin luxS_(V.h.) in V. harveyi eliminates AI-2 production.

Identification and analysis of S. typhimurium autoinducer productionmutants. In order to identify the gene responsible for AI-2 productionin S. typhimurium, we randomly mutagenized S. typhimurium LT2 using theMudJ transposon (Maloy et al., 1996, supra). Ten-thousand S. typhimuriumLT2 insertion mutants were assayed for AI-2 production in the V. harveyiBB170 bioassay. One S. typhimurium MudJ insertion mutant (strain CS132)was identified that lacked detectable AI-2 in culture fluids atmid-exponential phase.

FIG. 10B shows the AI-2 production phenotypes of S. typhimurium strainLT2 and the corresponding MudJ insertion strain CS 132. The strains weregrown to mid-exponential phase in LB containing glucose, and cell-freeculture fluids were prepared and assayed for AI-2. S. typhimurium LT2culture fluids induced the reporter strain 500-fold, while culturefluids from strain CS132 contained no AI-2 activity. Furthermore, strainCS132 did not produce AI-2 under any of the growth conditions that wehave previously reported induce AI-2 production in S. typhimurium (notshown).

The site of the MudJ insertion in S. typhimurium CS132 was determined byPCR amplification followed by sequencing of the 110 bp of chromosomalDNA adjacent to the transposon. This sequence was used to search thedatabase for DNA homologies. The sequence matched a site (89/105 bpidentity) in the E. coli MG1655 genome that corresponded to an ORF ofunknown function denoted ygaG (Blattner et al., 1997, supra). In thechromosome, the E. coli ygaG gene is flanked by the gshA and emrB genes(FIG. 9B). The ygaG gene is transcribed from its own promoter which islocated immediately upstream of the gene, indicating that it is not inan operon with gshA. The emrB gene is transcribed in the oppositedirection. We PCR amplified the ygaG region from the chromosomes of E.coli O157:H7 and E. coli MG1655, and the two E. coli ygaG genes werecloned into pUC19.

Complementation of S. typhimurium and E. coli AI-2⁻ mutants. We testedwhether the E. coli O157:H7ygaG gene and the V. harveyi luxS_(V.h.) genecould restore AI-2 production in the AI-2⁻ strains S. typhimurium CS132and E. coli DH5. In FIG. 11A, we show the AI-2 activity produced by wildtype V. harveyi BB120, E. coli O157:H7 and S. typhimurium LT2. In thisfigure, the level of AI-2 activity present in V. harveyi BB120 cell-freeculture fluids was normalized to 100%, and the activities in cell-freeculture fluids from E. coli and S. typhimurium compared to that. In thisexperiment, E. coli O157:H7 produced 1.5 times and S. typhimurium LT2produced 1.4 times more AI-2 activity than V. harveyi BB120 (i.e., 150%and 141% respectively).

FIGS. 11B and 11C show the AI-2 complementation results for S.typhimurium CS132 and E. coli DH5. FIG. 11B demonstrates thatintroduction of the E. coli O157:H7ygaG gene into S. typhimurium CS132restored AI-2 production beyond the level of production of wild type S.typhimurium (i e., 209% activity). Comparison of the data in FIGS. 11Aand 11B shows that the E. coli ygaG gene in S. typhimurium resulted inAI-2 production exceeding that produced in vivo by E. coli O157:H7.Introduction of the V. harveyi luxS_(V.h.) gene into S. typhimuriumresulted in AI-2 production at slightly less than the level produced bywild type V. harveyi BB120 (i.e., 73% of the level of V. harveyi BB120).FIG. 11C shows that E. coli DH5 was also complemented to AI-2 productionby both the cloned E. coli O157:H7 and the V. harveyi BB120 AI-2production genes. However, introduction of E. coli O157:H7ygaG and V.harveyi BB120 luxS_(V.h.) into E. coli DH5 resulted in only 31% and 43%of the V. harveyi BB120 AI-2 activity respectively. FIGS. 11B and 11Cshow that the control vectors produced no activity in thecomplementation experiments.

Analysis of the AI-2 production genes from V. harveyi, E. coli and S.typhimurium. We sequenced the AI-2 production gene luxS_(V. h.) from V.harveyi BB120 and the ygaG loci from E. coli O157:H7, E. coli MG1655 andE. coli DH5. The translated protein sequences encoded by the ygaG ORF'sare shown in FIG. 12, and they are aligned with the translated LuxSprotein sequence from V. harveyi. The non-bold, underlined amino acidsindicate the residues in the E. coli proteins that differ from the V.harveyi LuxS protein. The ygaG loci from E. coli encode proteins thatare highly homologous to one another and also to LuxS from V. harveyi.The E. coli MG1655 and the E. coli O157:H7 YgaG proteins are 77% and 76%identical to LuxS from V. harveyi BB120. The DNA sequence we determinedfor ygaG from E. coli O157:H7 differs at five sites from the reported(and our) sequence for the E. coli MG1655 ygaG gene. Four of the changesare silent, the fifth results in a conservative Ala to Val alteration atamino acid residue 103 in the E. coli O157:H7 protein.

Identification of the ygaG locus in E. coli MG1655 and E. coli O157:H7allowed us to investigate the AI-2 production defect in E. coli DH5. E.coli DH5 possesses the ygaG gene because we could PCR amplify thisregion from the chromosome using the same primers we employed to amplifyit from E. coli MG1655 and E. coli O157:H7. Examination of the E. coliDH5 ygaG promoter showed that it is identical to that of E. coli MG1655,indicating that the AI-2 defect in E. coli DH5 is not simply due todecreased transcription of ygaG. However, sequence analysis of the E.coli DH5 ygaG coding region showed that a one G-C base pair deletion anda T to A transversion exist at bp 222 and 224 respectively. Theframeshift mutation resulting from the G/C deletion causes prematuretruncation of the E. coli DH5 protein. FIG. 12 shows that the truncatedE. coli DH5 protein is 111 amino acids, while the E. coli MG1655 and E.coli O157:H7 proteins are 171 residues. Twenty altered amino acids aretranslated after the frame shift and prior to termination of theprotein. Our complementation results (FIG. 11) demonstrate that the AI-2production defect in E. coli DH5 is recessive to in trans expression ofygaG, which is consistent with the defect being due to a null mutationcaused by the frame shift in the E. coli DH5 ygaG gene.

We searched the S. typhimurium database using the sequence we obtainedadjacent to the MudJ that inactivated the AI-2 production function in S.typhimurium CS132. A perfect match (110/110 bp) was identified tofragment B_TR7095.85-T7 in the S. typhimurium LT2 genome sequencingdatabase (Genome Sequencing Center, Washington University, St. Louis).However, the S. typhimurium LT2 database ygaG sequence is incomplete(FIG. 12). The translated sequence matches the E. coli and V. harveyisequences beginning at amino acid residue 8. The translated sequenceshows that the S. typhimurium protein is 75% identical to LuxS of V.harveyi. In order to align the S. typhimurium sequence with the V.harveyi LuxS protein, we corrected three apparent frame shift errors inthe database sequence. Considering that only crude, unannotated sequencedata is currently available for S. typhimurium, we predict that the S.typhimurium protein contains 7 more amino acids, and that the frameshift mutations are sequencing errors. We were unsuccessful at PCRamplifying either the S. typhimurium 14028 or the S. typhimurium LT2ygaG gene using the primers designed for E. coli, so we do not yet havethe complete sequence of the S. typhimurium gene.

The results set forth above indicate that the genes we have identifiedand analyzed encode a novel family of proteins responsible forautoinducer production. Members of this new family of genes, referred toherein as LuxS, are highly homologous to one another but not to anyother identified genes. The encoded product of the LuxS genes catalyzean essential step in the synthesis of the signaling molecules of thepresent invention.

EXAMPLE 4 Construction of a Sensor 1⁻, AI⁻2⁻ V. harveyi Reporter Strain

V. harveyi null mutants in each of the lux genes luxL, luxM, luxN, luxSand luxQ have been constructed. These mutants fail to either make orrespond to one specific autoinducer, but they still produce lightbecause, in each case, one quorum sensing system remains operational. Adouble luxN, luxS V. harveyi mutant will not emit light without theaddition of exogenous AI-2 because this mutant will not respond to AI-1and it will not produce AI-2.

The V. harveyi luxS gene has been cloned into E. coli DH5α on a broadhost range mobilizable cosmid called pLAFR2. This construction restoresAI-2 production to E. coli DH5α. A marked null mutation was engineeredinto the luxS gene by introducing a chloramphenicol resistance (Cm^(r))cassette into an internal restriction site. Placement of the Cm^(r)cassette at this site in luxS subsequently eliminated AI-2 production inE. coli DH5α.

The luxS::Cm^(r) null allele was transferred onto the chromosome of V.harveyi strain BB170. Strain BB170 contains a Tn5Kan^(r) in luxN anddoes not respond to AI-1. To construct the double mutant, triparentalconjugations were carried out by mixing stationary phase cultures of E.coli DH5α carrying the V. harveyi luxS::CM^(r) construction in pLAFR2(pLAFR2 carries tetracycline resistance), E. coli DH5_carrying the tradonor plasmid pRK2013 and the V. harveyi recipient strain BB170.Exchange of the luxS::Cm^(r) mutant allele for the wild type luxS alleleon the chromosome occurs by homologous recombination. The exogenotecosmid in V. harveyi was eliminated by the introduction of a secondincompatible plasmid pPH1JI. This was accomplished by mating E. coliDH5α containing pPH1JI with the V. harveyi BB170 recipient containingthe luxS::Cm^(r) cosmid, and selecting for exconjugants on platescontaining ampicillin (for counter selection of the E. coli donor)chloramphenicol (for inheritance of the mutant luxS::Cm^(r) allele) andgentamicin (for maintenance of the plasmid pPH1JI). Southern blotanalysis was used to verify that the exogenote pLAFR2 cosmid has beeneliminated and that the luxS::Cm^(r) construction had been transferredto the corresponding position in the genome of V. harveyi. The pPH1JIcosmid was subsequently eliminated by growth in the absence ofgentamicin selection.

Verification that the luxN, luxS Double Mutant Responds to AI-2. The V.harveyi strain that is mutant in luxN and luxS was stimulated to producelight in response to the exogenous addition of AI-2 but not AI-1. Thiswas verified in a luminescence assay for response to V. harveyi AI-1 andAI-2. V. harveyi strain MM30 (luxS::Tn5) which is phenotypically AI-1⁺,AI-2⁻, and V. harveyi strain BB152 (luxM::Tn5) which is phenotypicallyAI-1⁻, AI-2⁺ were used as the sources of AI-1 and AI-2 respectively. TheAI-1 and AI-2 present in culture fluids of these strains was tested forstimulation of light production of the V. harveyi luxN, luxS doublemutant reporter strain. In this assay, autoinducer preparations fromMM30, BB152 or sterile medium controls were added to the wells ofmicrotiter plates, followed by the addition of the V. harveyi reporterstrain. The resulting light production was monitored using a liquidscintillation counter in the chemiluminescence mode. Maximal stimulationof light production in the V. harveyi luxN, luxS reporter strain wascompared to that produced by the Sensor 1⁺, Sensor 2⁻ V. harveyi strainBB886 and the Sensor 1⁻, Sensor 2⁺ V. harveyi strain BB170. These two V.harveyi strains are routinely used in this assay as reporters of AI-1and AI-2 activity respectively.

Determine optimum concentrations of AI-2 in microtiter assays. Theaformentioned screen will be optimized for use in 96-well microtiterassays. The screen will be used in inhibitor assays for identifyinginhibitors of AI-2. Purified or synthetic AI-2 will be added to themicrotiter wells containing the newly constructed reporter strain andinhibition will be measured by a decrease in light emission from thewells containing an inhibitor. The assay will be optimized bydetermining the concentration of cells and AI-2 in the microtiter wellsthat will allow for maximal sensitivity. The optimal AI-2 concentrationwill be that which stimulates half-maximal light output for a givenconcentration of cells per unit time. Initial experiments will beconducted in this concentration range to determine the range of AI-2concentration that produces the greatest change in light output. Similarexperiments using AI-1 and a non self-stimulating sensor-1⁺, sensor-2⁻mutant (BB886) showed that the assay was sensitive to concentrations aslow as 100 nM AI-1 and that light emission was linear over 6 orders ofmagnitude (light emission from a self-stimulating strain was linear over3 orders). Similar results for AI-2 using the new reporter strain whichwill not self-stimulate and therefore have zero background lightemission are predicted. Light emission from the microtiter wells will bemeasured with a Wallac TriLux liquid scintillation counter model1450-021 in the chemiluminescence mode. This machine will accommodate 16plates and will therefore allow for 1536 separate concentrationexperiments per run.

EXAMPLE 5 In-Vitro Method for Synthesizing AI-2

Purification and Identification of AI-2. The AI-2 class of molecule isrefractory to purification by conventional techniques used for theisolation of acyl-homoserine lactone (HSL) autoinducers such as AI-1from V. harveyi. Unlike other HSL autoinducers, under the conditionstested, the AI-2 activity does not extract quantitatively into organicsolvents. Furthermore, it fails to bind to either a cation or anionexchange column. The present characterization of AI-2 indicates that ithas a molecular weight of less than 1000 kDa, and is a polar butapparently uncharged organic compound. The AI-2 activity is acid stableand base labile and heat resistant to about 80 but not 100° C. Theseresults indicate that the AI-2's are not acyl-homoserine lactones. TheluxS genes identified in the present study bear no homology to othergenes known to be involved in production of HSL autoinducers furtherindicating that the present AI-2 class of autoinducers is novel.

Thus, in addition to providing a cloned, overexpressed and purified S.typhimurium LuxS protein, the present invention also provides a methodfor producing AI-2 in vitro. The present invention provides a mechanismfor generating large quantities of pure AI-2 useful for mass spectraland NMR analysis, and for screening compounds which modulate theactivity of AI-2. Moreover, the present invention provides a method fordetermining the in vivo biosynthetic pathway for AI-2 synthesis.

The analysis of the genomic locations of the various luxS genesidentified in the present invention indicates that the luxS genes do notconsistently reside in any one location in the chromosome, nor are theytypically found in close proximity to any specific gene(s). However, inone case, the luxS gene is the third gene in a three-gene operon withtwo genes (metK and pfs). In E. coli, Salmonella and many otherbacteria, MetK and Pfs are involved in the conversion of S-adenosylmethionine (SAM) to homocysteine and 4,5-dihydroxy-2,3 pentanedione(FIG. 15). The function of MetK is to convert methionine to SAM which isan important cofactor in one-carbon metabolism. SAM is used to methylateDNA, RNA and a variety of cell proteins, and several SAM dependentmethyl transferases act at this step. S-adenosyl homocysteine (SAH) isproduced when the methyl group is transferred from SAM to itssubstrates. SAH functions as a potent inhibitor of SAM dependentmethyltransferases. Therefore, bacteria rapidly degrade SAH via theenzyme Pfs. The designation “pfs” refers to an open reading frame in theE.coli genome that has recently been determined to encode the enzyme5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase, also knownas MTA/SAH nucleosidase. In the present system, the enzyme cleaves theglycosidic bond in S-adenosylhomocycteine (SAH). Thus, the function ofPfs is to convert SAH to adenine and S-ribosyl homocysteine. In a finalstep, S-ribosyl homocysteine is converted to homocysteine and4,5-dihydroxy-2,3-pentanedione. Homocysteine can re-enter this pathway;it is methylated to generate methionine which can be converted to SAM byMetK.

The catabolism of SAH is considered a salvage pathway for recyclingmetabolic intermediates (adenine and homocysteine). However, somespecies of bacteria eliminate SAH by a different pathway. In thisalternative pathway, adenosine is directly removed from SAH whichgenerates homocysteine. Therefore, cells that use this second mechanismdo not produce 4,5-dihydroxy-2,3-pentanedione. In the pathway shown inFIG. 15, the enzyme responsible for conversion of S-ribosyl homocysteineto 4,5-dihydroxy-2,3-pentanedione has never been identified, cloned orpurified. Furthermore, no role for 4,5-dihydroxy-2,3-pentanedione isknown.

LuxS is involved in the pathway shown in FIG. 15, and SAM and SAH areinvolved in AI-2 production. The structure of AI-2 could be4,5-dihydroxy-2,3-pentanedione, in which case LuxS is theuncharacterized enzyme that acts on S-ribosyl homocysteine. Second, LuxScould act on one of the intermediates to make AI-2. LuxS would representa branch point off the known pathway.

To confirm that LuxS is involved in the conversion of SAM to AI-2, thegene encoding the S. typhimurium LuxS protein was cloned, overexpressedand the S. typhimurium LuxS protein was purified. This protein was usedin combination with dialyzed cell-free extracts prepared from a S.typhimurium luxS null mutant to show that addition of SAM and LuxSprotein could restore AI-2 production to dialyzed LuxS-cell extracts.Reaction mixtures were prepared containing 10 mM Sodium Phosphate bufferpH 7.0, dialyzed S. typhimurium LuxS-cell extract and SAM. Purified LuxSprotein was added to some of these mixtures. The reactions wereincubated at room temperature for 60 min, followed by centrifugation ina 5000 MWCO centricon. The material with MW<5000 was added to thestandard V. harveyi bioassay as previously described. Dialyzed LuxS-cellextracts to which SAM was added or extracts containing LuxS proteinwithout the addition of SAM produced no AI-2 activity. However,identical extracts to which LuxS protein and SAM had been added producedAI-2 that resulted in over 500-fold stimulation in light production inthe bioassay.

Further investigation showed that SAM is not the direct substrate forLuxS, and that LuxS must act at a step subsequent to the conversion ofSAM to SAH (FIG. 15). It was determined that AI-2 was not produced ifSAM was added directly to LuxS protein, however activity was produced bypre-incubation of SAM with the LuxS-extracts, filtration, and subsequentaddition of LuxS protein to the filtrate. Importantly, these studiesindicate that SAM can react with an element in the cell extract beforeit can be used by LuxS to make AI-2. Presumably, the SAM dependentmethyl transferases present in the cell extract use SAM as a methyldonor and convert it to SAH in the process. To verify this, SAH wassubstituted for SAM in an in vitro assay. Addition of SAH to the invitro assay resulted in much greater AI-2 production than when SAM wasadded. This result indicates that LuxS functions in the pathwaysubsequent to the conversion of SAM to SAH. Again, addition of SAHdirectly to LuxS protein is not sufficient for production of AI-2activity, while pre-incubation of SAH with dialyzed LuxS-extractsfollowed by filtration and subsequent addition of LuxS protein to thefiltrates does result in AI-2 production. Presumably SAH is converted toS-ribosyl homocysteine and then LuxS acts to produce AI-2.

The proposed pathway shown in FIG. 15 is not a salvage pathway forrecycling secondary metabolites, but rather is the pathway forproduction of AI-2. The present invention has narrowed the possibilitiesfor point of LuxS activity in the biosynthesis of AI-2. The remainingpossibilities are shown in FIG. 15 (designated LuxS?).

According to the invention, AI-2 is a derivative of ribose. It isnoteworthy that, in V. harveyi, LuxP, the primary sensor for AI-2, is ahomologue of the E. coli and S. typhimurium ribose binding protein.

Characterization and Biosynthesis of AI-2. The invention furtherprovides a method for an in vitro procedure for large scale productionof pure AI-2. As indicated in FIG. 15, SAH is a precursor in the LuxSdependent biosynthesis of AI-2. Furthermore, LuxS does not act directlyon SAH. Prior to the action of LuxS, SAH must first be acted on by someenzyme in dialyzed cell extracts. Presumably this step is the conversionof SAH to S-ribosyl homocysteine by Pfs. Therefore the substrate forLuxS is S-ribosyl homocysteine.

To confirm that LuxS acts on S-ribosyl homocysteine, the Pfs enzyme canbe purified and used to convert SAH to S-ribosyl homocysteine. Towardthis end, the pfs gene has been cloned from S. typhimurium 14028 placedinto the overexpression vector pLM-1. The Pfs enzyme will beoverexpressed and SAH will be added to purified Pfs to produce S-ribosylhomocysteine. The conversion of SAH to S-ribosyl homocysteine will beconfirmed by reverse phase HPLC analysis (SAH is UV active whileS-ribosyl homocysteine is not). Subsequently, the S-ribosyl homocysteineproduced by Pfs will be added to purified LuxS. Following incubation,the mixture will be filtered over a 5000 MWCO centricon. The filtratewill be tested for AI-2 activity in the previously described V. harveyibioassay. The identification of activity will confirm that4,5-dihydroxy-2,3-pentanedione is AI-2.

In addition, AI-2 structure obtained from E. coli and V. harveyi AI-2will be determined. The E. coli and V. harveyi luxS genes have beencloned in to overexpression vectors. The identity/biosynthesis of the S.typhimurium AI-2 provided by the present invention should greatlyfacilitate these analyses. In the event that the S. typhimurium, E. coliand V. harveyi AI-2's are identical these data will indicate that AI-2'sare the same.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. An isolated bioluminescent Vibrio harveyi bacterial strain comprisinga first genetic alteration in the LuxN gene that inhibits thebioluminescence of the Vibrio harveyi bacterial strain in response toautoinducer-1 of Vibrio harveyi and a second genetic alteration in theLuxS gene that inhibits production of a autoinducer-2 of Vibrio harveyi.2. A kit comprising the Vibrio harveyi bacterial strain of claim
 1. 3.The bacterial strain of claim 1, wherein the second genetic alterationinhibits production of a pentanedione.
 4. The bacterial strain of claim3 wherein the pentanedione is 4,5-dihydroxy-2,3-pentanedione.
 5. The kitof claim 2, wherein the second genetic alteration inhibits production ofa pentanedione.
 6. The kit of claim 5 wherein the pentanedione is4,5-dihydroxy-2,3-pentanedione.